Multiphoton microscopy in life sciences

Near infrared (NIR) multiphoton microscopy is becoming a novel optical tool of choice for fluorescence imaging with high spatial and temporal resolution, diagnostics, photochemistry and nanoprocessing within living cells and tissues. Three‐dimensional fluorescence imaging based on non‐resonant two‐photon or three‐photon fluorophor excitation requires light intensities in the range of MW cm^?2 to GW cm^?2, which can be derived by diffraction limited focusing of continuous wave and pulsed NIR laser radiation. NIR lasers can be employed as the excitation source for multifluorophor multiphoton excitation and hence multicolour imaging. In combination with fluorescence in situ hybridization (FISH), this novel approach can be used for multi‐gene detection (multiphoton multicolour FISH). Owing to the high NIR penetration depth, non‐invasive optical biopsies can be obtained from patients and ex vivo tissue by morphological and functional fluorescence imaging of endogenous fluorophores such as NAD(P)H, flavin, lipofuscin, porphyrins, collagen and elastin. Recent botanical applications of multiphoton microscopy include depth‐resolved imaging of pigments (chlorophyll) and green fluorescent proteins as well as non‐invasive fluorophore loading into single living plant cells. Non‐destructive fluorescence imaging with multiphoton microscopes is limited to an optical window. Above certain intensities, multiphoton laser microscopy leads to impaired cellular reproduction, formation of giant cells, oxidative stress and apoptosis‐like cell death. Major intracellular targets of photodamage in animal cells are mitochondria as well as the Golgi apparatus. The damage is most likely based on a two‐photon excitation process rather than a one‐photon or three‐photon event. Picosecond and femtosecond laser microscopes therefore provide approximately the same safe relative optical window for two‐photon vital cell studies. In labelled cells, additional phototoxic effects may occur via photodynamic action. This has been demonstrated for aminolevulinic acid‐induced protoporphyrin IX and other porphyrin sensitizers in cells. When the light intensity in NIR microscopes is increased to TW cm^?2 levels, highly localized optical breakdown and plasma formation do occur. These femtosecond NIR laser microscopes can also be used as novel ultraprecise nanosurgical tools with cut sizes between 100 nm and 300 nm. Using the versatile nanoscalpel, intracellular dissection of chromosomes within living cells can be performed without perturbing the outer cell membrane. Moreover, cells remain alive. Non‐invasive NIR laser surgery within a living cell or within an organelle is therefore possible.     

Two-photon excited lifetime imaging of autofluorescence in cells during UV A and NIR photostress

By monitoring coenzyme autofluorescence modifications. as an indicator of cell damage. the cellular response to femtosecond near-infrared (NIR) radiation (two-photon absorption) was compared with exposure to low-power UV A radiation (one-photon absorption). Excitation radiation from a tunable Ti-sapphire laser. focused through highnumerical- aperture microscope optics. provided diffractionlimited mlcrobeams of an adjustable peak power. Laser scanning NIR microscopy was used to detect spatially the intracellular distribution of fluorescent coenzymes by fluorescence intensity imaging as well as fluorescence lifetime imaging (_T-mapping). Upon the onset of UV or NIR exposure. Chinese hamster ovary cells exhibited blue/green autofluorescence witq a mean lifetime of 2·2 ns. which was attributed to NAD(P)H in mitochondria. Exposure to 365 nm radiation from a high-pressure mercury lamp (1 m W. 300 J cm^-2 ) resulted in oxidative stress correlated with increased autofluorescence intensity. onset of nuclear fluorescence. and a fluorescence lifetime decrease. The cellular response to femtosecond NIR micro beams depended significantly on peak power. Peak powers above a threshold value of about 0·5kW (average power: 6mW). 0·55kW (7mW) and 0·8kW (lOmW) at 730nm. 760nm and 800nm. respectively. resulted in the onset of short-lived luminescence with higher intensity (100x) than the intracellular NAD(P)H fluorescence. This luminescence. accompanied by destruction of cellular morphology. was localized and occurred in the mitochondrial region. In contrast. beams at a power of less than 0·5 kW allowed nondestructive fluorophore detection with high spatial and temporal resolution without modification of cellular redox state or cell morphology.     

Fluorescence photobleaching recovery in the confocal scanning light microscope

Measurement of mobilities of species in liquid systems is of great importance for understanding a number of dynamic phenomena. A well known method for measuring mobilities driven by diffusion is fluorescence photobleaching recovery (FPR), also known as fluorescence recovery after photobleaching (FRAP). New FPR recovery equations for three-dimensional (3-D) apertured scanning using a Gaussian approximation for the axial beam profile have been successfully developed and found to provide a solid basis for extraction of the lateral diffusion coefficient from confocal scanning light microscopy (CSLM)-FPR experimental data. The 2-D diffusion coefficients of fluorescent species can be successfully measured by FPR in the CSLM, which has the great advantage that bleaching can be targeted at a well-defined volume element in bulk samples. Two-dimensional diffusion coefficients of 45-nm latex spheres, of FITC molecules and of a 2·45-nm protein-FITC complex in water-glycerol mixtures, measured by FPR in the CSLM, are in close agreement with those calculated from the size of the diffusing species and viscosity of the medium. Diffusion coefficients as high as 2 times 10^?6 cm²/s can be measured.     

Visualization of the native shape of bodipy‐labeled DNA in Escherichia coli by correlative microscopy

The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL‐14‐dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high‐resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 ～ 10 mmol/kg in area of 0.1 μm², which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase‐plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase‐plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.     

An infra-red light-transmitting aperture controller for use in single-cell fluorescence photometry

Photometric techniques are commonly used to monitor the output from fluorescent indicators during the study of cellular signalling. At the single-cell level, the region of interest is normally set by a variable aperture placed within the microscope emission pathway. The present study reports an improved aperture controller which adjusts the area for fluorescence measurement, whilst allowing objects throughout the field of view to be continuously monitored using infra-red illumination. A rectangular aperture is selected by four 715-nm long-pass glass filters which block > 99.9% of the fluorescence emission at 480–600 nm. A 780-nm long-pass glass filter is used to provide infra-red illumination which does not interfere with the fluorescence signal, yet is detectable by a standard CCD camera. This allows detection of morphological events throughout the field of view and facilitates manipulation of extracellular pipettes, without interruption to a single-cell fluorescence recording. The infra-red light-transmitting controller is suitable for use with a range of other fluorescent indicators, including those routinely used to detect Ca²⁺, Cl^?, Na⁺ and pH. Data are presented which demonstrate the use of this controller to measure ADP-evoked [Ca²⁺]_i increases in single human erythroleukaemia cells loaded with the Ca²⁺ indicator fura-2.     

Variable-angle total internal reflection fluorescence microscopy (VA-TIRFM): realization and application of a compact illumination device

A novel compact illumination device in variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) is described. This device replaces the standard condensor of an upright microscope. Light from different laser sources is delivered via a monomode fibre and focused onto identical parts of a sample under variable angles of total internal reflection. Thus, fluorophores in close proximity to a cell–substrate interface are excited by an evanescent wave with variable penetration depth, and localized with high (nanometre) axial resolution. In addition to quantitative measurements in solution, fluorescence markers of the cytoplasm and the plasma membrane, i.e. calcein and laurdan, were examined using cultivated endothelial cells. Distances between the glass substrate and the plasma membrane were determined using the mathematical algorithm of a four‐layer model, as well as a Gaussian‐shaped intensity profile of the illumination spot on the samples. Distances between 0 and 30 nm in focal contacts and between 100 and 300 nm in other parts of the cell were thus determined. In addition to measurements of cell–substrate topology, the illumination device appears appropriate for numerous applications in which high axial resolution is required, e.g. experiments on endocytosis or exocytosis, as well as measurements of ion concentrations proximal to the plasma membrane. The compact illumination device is also suitable for combining TIRFM with further innovative techniques, e.g. time‐resolved fluorescence spectroscopy, fluorescence lifetime imaging (FLIM) or fluorescence resonance energy transfer (FRET).     

A design for a simple electronic exposure meter for use with an electron microscope

A circuit has been developed to enable a simple yet sensitive exposure meter to be made for any electron microscope. The circuit is based around an inexpensive operational amplifier and will indicate a current range 10 ^?4?10 ^?11 A which corresponds from maximum illumination to a level of fluorescent screen illumination which is not detectable by eye. The exposure meter measures the charge on the fluorescent screen which is correlated to exposure time for photography.     

Specific labeling of connexin43 in NRK cells using tyramide-based signal amplification and fluorescence photooxidation

Imaging of gap junction proteins, the connexins, has been performed in tissue culture cells both by labeling of connexins with immunocytochemical tags and by cloning and expressing chimeras of connexins and fluorescent proteins such as Green Fluorescent Protein. These two approaches have been used to gain information about protein localization or trafficking at light microscopic resolution. Electron microscopy provides higher resolution; however, analysis of electron micrographs of unlabeled connexins has been generally limited to recognition of gap junction structures. Immunolabeling of gap junction proteins in whole cells at the electron microscopic level has been difficult to achieve because of the fixation sensitivity of most gap junction antibodies. To obtain reasonable sensitivity, immunoperoxidase procedures are typically employed, and these suffer from relatively poor resolution. Here we describe the combination of tyramide signal amplification techniques and fluorescence photooxidation for higher resolution immunolocalization studies for correlative light and electron microscopic imaging. By using correlative microscopy, we can not only localize connexin pools or structures, but also discover what other cellular substructures interact with gap junction proteins. The use of tyramide signal amplification techniques is necessary to increase fluorescence levels that have decreased due to increased specimen fixation required to maintain cell ultrastructure. The fluorescence photooxidation technique provides a high-resolution method for staining of proteins in cells. Unlike colloidal gold-based methods, fluorescence photooxidation allows for three-dimensional localization using high-voltage electron microscopy.     

Phase separation as a basis for the formation of light-emitting silicon nanoclusters in SiO x films irradiated with swift heavy ions

This paper presents a study of the effect of swift heavy Xe ions of energy 130–167 MeV at doses of 10¹²–10¹⁴ cm^?2 and Bi ions of 700 MeV at doses of 3·10¹²–3·10¹³ cm^?2 on films of stoichiometric thermal silicon dioxide, silicon dioxide films with ion-implanted excess silicon, and SiO _x films with the stoichiometric parameter x varying from 0 to 2. According to electron microscopy and Raman spectroscopy data, irradiation with the swift heavy ions resulted in the formation of silicon nanoclusters. The luminescence spectra depended on the size, number, and structure of the Si nanoclusters formed. Their size can be controlled by varying both the effect parameters (primarily, the ion energy loss per unit length of the track) and the stoichiometric composition of the films.     

Quantitative analysis of mast cell structure

Fixed and embedded normal rat peritoneal mast cells were studied by light and electron microscopy, utilizing stereological methods to obtain quantitative data on their structure. The diameters of the mast cells and their nuclei averaged 10.9 and 5.8 μm respectively. The volume of the individual mast cell granule was estimated to 0.3 μm³. About 53% of the cytoplasmic volume was occupied by granules, and 2% by mitochondria. 11% of the cell volume was taken up by the nucleus. The average number of granules per cell was calculated to about 1020. Quantitative biochemical data on mast cells, extrapolated from the literature and applied to the calculated figures above, yield the following results with respect to mast granule contents-heparin: 95 × 10^?3 pg, histamine: 30 × 10^?3 pg and 5-hydroxytryptamine: 1.3 × 10^?3 pg per mast cell granule.     

Tracking of secretory vesicles of PC12 cells by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy is used to detect cellular events near the plasma membrane. Behaviours of secretory vesicles near the cell surface of living PC12 cells, a neuroendocrine cell line, are studied. The secretory vesicles are labelled by over‐expression of enhanced green fluorescent protein‐tagged Rab3A, one of the small G proteins involved in the fusion of secretory vesicles to plasma membrane in PC12 cells. Images acquired by a fast cooled charge‐coupled device camera using conventional fluorescence microscopy and total internal reflection fluorescence microscopy are compared and analysed. Within the small evanescent range (< 200 nm), the movements of the secretory vesicles of PC12 cells before and after stimulation by high K⁺ are examined. The movements of one vesicle relative to another already docked on the membrane are detected. Total internal reflection fluorescence microscopy provides a novel optical method to trace and analyse the exocytotic events and vesicle specifically near a cell membrane without interference of signals from other parts of the cell.     

Ultrastructural effects of two phthalocyanines in CHO-K1 and HeLa cells after laser irradiation

The effects of Photodynamic Therapy using 2^nd generation photosensitizers have been widely investigated aiming clinical application treatment of solid neoplasms. In this work, ultrastructure changes caused by the action of two 2^nd generation photosensitizers and laser irradiation on CHO-K1 and HeLa (neoplastic) cells were analyzed by transmission electron microscopy. Aluminum phthalocyanine chloride, aluminum phthalocyanine tetrasulfonate chloride and radiation from a semiconductor laser at a fluency of 0.5 J/ cm² (Power=26mW; λ=670nm) were used. The results showed induction of apoptosis. Such alterations where observed in HeLa but not in CHO-K1 cells after Aluminum phthalocyanine tetrasulfonate chloride (AlPcS₄) photodynamic treatment. The Aluminum phthalocyanine chloride (AlPc) photodynamic treatment induced necrosis on the neoplastic cell line, and cytoplasm and nuclear alterations on the normal cell line.     

Metabolic labeling of Escherichia coli genomic DNA with erythrosine‐11‐dUTP for functional imaging via correlative microscopy

The fluorescent metabolic labeling of microorganisms genome is an advanced imaging technique to observe and study the native shapes, structural changes, functions, and tracking of nucleic acids in single cells or tissues. We have attempted to visualize the newly synthesized DNA within the intact nucleoid of ice‐embedded proliferating cells of Escherichia coli K‐12 (thymidine‐requiring mutant, strain N4316) via correlative light‐electron microscopy. For that purpose, erythrosine‐11‐dUTP was synthesized and used as a modified analog of the exogenous thymidine substrate for metabolic incorporation into the bacterial chromosome. The formed fluorescent genomic DNA during in cellulo polymerase reaction caused a minimal cellular arrest and cytotoxicity of E. coli at certain controlled conditions. The stained cells were visualized in typical red emission color via an epifluorescence microscope. They were further ice‐embedded and examined with a Hilbert differential contrast transmission electron microscopy. At high‐resolution, the ultrastructure of tagged nucleoid appeared with significantly higher electron dense in comparison to the unlabeled one. The enhanced contrast areas in the chromosome were ascribed to the presence of iodine contents from erythrosine dye. The presented labeling approach might be a powerful strategy to reveal the structural and dynamic changes in natural DNA replication including the relationship between newly synthesized in vivo nucleic acid and the physiological state of the cell.     

Comparative effects of two different doses of low‐level laser therapy on wound healing third‐degree burns in rats

Burns are injuries caused by direct or indirect contact to chemical, physical, or biological agents. Low‐level laser therapy (LLLT) is a promising treatment since it is low‐cost, non‐invasive, and induces cell proliferation. This study aimed to investigate the effects of LLLT (660 nm) at two different fluences (12.5 J/cm² and 25 J/cm²) per point of application on third‐degree burns in rats. Thirty rats (Wistar) divided into GC, GL12.5, and GL25 were used in the study, and submitted to burn injury through a soldering iron at 150°C, pressed on their back for 10 s. LLLT was applied immediately, and 2, 4, 6, and 8 days after wound induction. Histological analysis revealed a decreased inflammatory infiltrate in the group treated with 25 J/cm², and intense inflammatory infiltrate in the control group and in the group treated with 12.5 J/cm². The immunostaining of COX‐2 was more intense in the control groups and in the group treated with 12.5 J/cm² than in the group treated with 25 J/cm². Conversely, VEGF immunomarking was more expressive in the group treated with 25 J/cm² than it was in the other two groups. Therefore, our findings suggest that the use of 25 J/cm² and 1 J of energy was more effective in stimulating the cellular processes involved in tissue repair on third‐degree burns in rats by reducing the inflammatory phase, and stimulating angiogenesis, thus restoring the local microcirculation which is essential for cell migration. Microsc. Res. Tech. 79:313–320, 2016. © 2016 Wiley Periodicals, Inc.     

Effect of Er:YAG laser irradiation on deciduous enamel roughness and bacterial adhesion: An in vitro study

Laser irradiation has been proposed as a preventive method against dental caries since it is capable to inhibit enamel demineralization by reducing carbonate and modifying organic matter, yet it can produce significant morphological changes. The purpose of this study was to evaluate the influence of Er:YAG laser irradiation on superficial roughness of deciduous dental enamel and bacterial adhesion. Fifty‐four samples of deciduous enamel were divided into three groups (n = 18 each). G1_control (nonirradiated); G2_100 (7.5 J/cm²) and G3_100 (12.7 J/cm²) were irradiated with Er:YAG laser at 7.5 and 12.7 J/cm², respectively, under water irrigation. Surface roughness was measured before and after irradiation using a profilometer. Afterwards, six samples per group were used to measure bacterial growth by XTT cell viability assay. Adhered bacteria were observed using confocal laser scanning microscopy (CLSM) and a scanning electron microscopy (SEM). Paired t‐, one‐way analysis of variance (ANOVA), Kruskal‐Wallis and pairwise Mann–Whitney U tests were performed to analyze statistical differences (p < .05). Before treatment, samples showed homogenous surface roughness, and after Er:YAG laser irradiation, the surfaces showed a significant increase in roughness values (p < .05). G3_100 (12.7 J/cm²) showed the highest amount of Streptococcus mutans adhered (p < .05). The increase in the roughness of the tooth enamel surfaces was proportional to the energy density used; the increase in surface roughness caused by laser irradiation did not augment the adhesion of Streptococcus sanguinis; only the use of the energy density of 12.7 J/cm² favored significantly the adhesion of S. mutans.     

Imaging and measurement of cytosolic free calcium in plant and fungal cells

Calcium plays a central role as a second messenger in plant and fungal cells and as such is involved in controlling numerous biological processes. Direct demonstration of signal-response coupling via Ca²⁺ requires the measurement and localization of changes in cytosolic free Ca²⁺, [Ca²⁺]_i, during these processes in living cells. In recent years this has become possible with the introduction of a range of fluorescent dyes (e.g. Indo-1 Fura-2 and Fluo-3) which have a high affinity and selectivity for free Ca²⁺. When used with recently developed microscope technologies (e.g. fluorescence ratio imaging or confocal scanning laser microscopy), subcellular localization and precise quantification of [Ca²⁺]_i dynamics in single cells can be achieved. This review describes the principles of [Ca²⁺]_i imaging and measurement using fluorescent dyes, the equipment required to do it, the problems with botanical material and how they are being overcome, future developments for this approach in plant cell biology, and an entirely different strategy for the imaging and measurement of [Ca²⁺]_i involving genetic transformation with the aequorin gene.     

Label‐free optical detection of age‐related and diabetic oxidative damage in human aqueous humors

In this study, we investigate the biochemical characteristics of oxidative stress in age‐related macular degeneration (AMD) and diabetic retinopathy (DR) by analyzing aqueous humors. Nondiabetic cataract aqueous humor was used as the control. The level of oxidative damage was evaluated based on changes in Raman spectral intensity. Seven prominent peaks were detected at 1002, 1043, 1062, 1352, 1419, 1454, and 1656 cm^?1. We proposed four multimodal biomarkers to distinguish these peaks based on the ratios of Raman intensities in two wavelengths, including CHO (C–O stretching or C–O–H bending modes), AG (adenine and guanine), PRO‐AG (protein and AG), and PHEα (phenylalanine symmetric ring breath and amide I α‐helix) markers. The presence of oxidative damage was detected by CHO and AG markers associated with C–O stretching, C–O–H bending modes in carbohydrates (1043 cm^?1), and the nucleic acids adenine and guanine (1352 cm^?1), respectively. DR‐related oxidative damage was identified by PRO‐AG and PHEα markers associated with adenine, guanine, and protein components (1419 and 1454 cm^?1) and amide I α‐helix protein structure (1656 cm^?1), respectively. AMD‐related oxidative damage was identified by four biomarkers. Four multimodal biomarkers with simple linear threshold values achieved high sensitivity of 100% and high specificity of 100% for classifying oxidative stress‐induced AMD and DR diseases. Therefore, Raman‐based label‐free optical detection is effective for detecting the presence of age‐related or diabetic oxidative damage in aqueous humor.     

Light scattering microscopy with angular resolution and its possible application to apoptosis

An inverted microscope has been modified for light scattering experiments with high angular resolution in combination with transmission, wide‐field fluorescence or laser scanning microscopy. Supported by simulations of Mie scattering, this method permits detection of morphological changes of 3T3 fibroblasts on apoptosis and formation of spherically shaped cells of about 20 μm diameter, in agreement with visual observation. Smaller sub‐structures (e.g. cell nuclei) as well as cell clusters may possibly contribute to the scattering behaviour. Results of 2‐dimensional cell cultures are confirmed by 3‐dimensional multicellular spheroids of 3T3 fibroblasts and HeLa 2E8 cervix carcinoma cells, where in most cases no morphological changes are discernable. This offers some advantage of light scattering microscopy for label‐free detection of apoptosis and may represent a first step towards label‐free in vivo diagnostics.     

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK₁ cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144 , 15–37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K⁺, Na⁺ and Ca⁺). Structurally damaged cells revealed lower K⁺ and higher Na⁺ and Ca⁺ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.     

Contrast effects in photoelectron microscopy; UV dose-dependent quantum yields of biological surface components

The relative brightness of photoelectron microscopy images as a function of exposure to UV light has been determined from model systems representative of biological cell surface components. Quantitative data for amino acid homopolymers, L-α-dipalmitoylphosphatidylcholine, and the polysaccharide Ficoll are reported as the absolute photoelectron quantum yields. The photoelectron quantum yields increase substantially over the initial values. For example, the quantum yields of poly-L-tyrosine at 200 nm is initially about 5 × 10^?8 electrons/incident photon. The quantum yield increases with 254 nm irradiation, leveling off at about 5 × 10^?4 electrons/incident photon after a dose of 3 × 10²¹ quanta cm^?2. Pre-irradiation of poly-L-tyrosine in the presence of certain chemical agents, for example, the Lewis base diborane (B₂H₆), results in a substantial reduction of the dose-dependent increase in quantum yield. Exposure to the reducing agent stannane (SnH₄) essentially eliminates the effect. These chemical treatments provide methods of controlling the UV dose-dependent effects in the photoelectron images.