Label‐free identification of antibiotic resistant isolates of living Escherichia coli: Pilot study

We introduce a label‐free spectroscopic method to classify subtypes of quinolone‐nonsusceptible Escherichia coli (E. coli ) isolates obtained from human blood cultures. Raman spectroscopy with a 30‐nm gold‐deposited, surface‐enhanced Raman scattering (SERS) substrate was used to evaluate three multilocus sequencing typing (MLST)‐predefined groups including E . coli ATCC25922, E . coli ST131:O75, and E . coli ST1193:O25b. Although there was a coffee‐ring effect, the ring zone was selected at the ideal position to screen E. coli isolates. Strong Raman peaks were present at 1001–1004 cm^?1 (C? C aromatic ring breathing stretching vibrational mode of phenylalanine), 1447–1448 cm^?1 (C? H₂ scissoring deformation vibrational mode), and 1667 cm^?1 (amide I α‐helix). Although the three MLST‐predefined E . coli isolates had similar Raman spectral patterns, a support vector machine (SVM) learning algorithm‐assisted principal component analysis (PCA) analysis had superior performance in detecting the presence of quinolone‐nonsusceptible E. coli isolates as well as classifying similar microbes, such as quinolone‐nonsusceptible E . coli ST131:O75 and E . coli ST1193:O25b isolates. Therefore, this label‐free and nondestructive technique is likely to be useful for clinically diagnosing quinolone‐nonsusceptible E. coli isolates with the MLST method.     

Label‐free monitoring of inflammatory tissue conditions using a carrageenan‐induced acute inflammation rat model

Although the confirmation of inflammatory changes within tissues at the onset of various diseases is critical for the early detection of disease and selection of appropriate treatment, most therapies are based on complex and time‐consuming diagnostic procedures. Raman spectroscopy has the ability to provide non‐invasive, real‐time, chemical bonding analysis through the inelastic scattering of photons. In this study, we evaluate the feasibility of Raman spectroscopy as a new, easy, fast, and accurate diagnostic method to support diagnostic decisions. The molecular changes in carrageenan‐induced acute inflammation rat tissues were assessed by Raman spectroscopy. Volumes of 0 (control), 100, 150, and 200 µL of 1% carrageenan were administered to rat hind paws to control the degree of inflammation. The prominent peaks at [1,062, 1,131] cm^?1 and [2,847, 2,881] cm^?1 were selected as characteristic measurements corresponding to the C–C stretching vibrational modes and the symmetric and asymmetric C–H (CH₂) stretching vibrational modes, respectively. Principal component analysis of the inflammatory Raman spectra enabled graphical representation of the degree of inflammation through principal component loading profiles of inflammatory tissues on a two‐dimensional plot. Therefore, Raman spectroscopy with multivariate statistical analysis represents a promising method for detecting biomolecular responses based on different types of inflammatory tissues.     

Flexible and stable optical parametric oscillator based laser system for coherent anti‐Stokes Raman scattering microscopy

The characteristics of a stable and flexible laser system based on a synchronously pumped optical parametric oscillator (OPO) is presented. This OPO can offer very stable operation with both ～1 ps and ～300 fs outputs over a broad wavelength range, i.e., 920–1200 nm. Combining the pump tuning with the OPO tuning, a total Raman range of 1900–5500 cm^?1 is accessible. For maximum spectral sensitivity, the CARS microsope based on the ps laser system is presented in detail. The lateral resolution of the microscope is diffraction limited to be about 390 nm. Fast wavelength switching (sub‐second) between two Raman vibrational frequencies, i.e., 2848 cm^?1 for C? H aliphatic vibrations and 3035 cm^?1 for C? H aromatic vibrations is presented as an example, although this also extends to other Raman frequencies. The possibility of obtaining a multimodal imaging system based on the fs laser system is also discussed. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Two‐frequency CARS imaging by switching fiber laser excitation

To fully exploit the power of coherent Raman imaging, techniques are needed to image more than one vibrational frequency simultaneously. We describe a method for switching between two vibrational frequencies based on a single fiber‐laser source. Stokes pulses were generated by soliton self‐frequency shifting in a photonic crystal fiber. Pump and Stokes pulses were stretched to enhance vibrational resolution by spectral focusing. Stokes pulses were switched between two wavelengths on the millisecond time scale by a liquid‐crystal retarder. Proof‐of‐principle is demonstrated by coherent anti‐Stokes Raman imaging of polystyrene beads embedded in a poly(methyl methacrylate) (PMMA) matrix. The Stokes shift was switched between 3,050 cm^?1, where polystyrene has a Raman transition, and 2,950 cm^?1, where both polystyrene and PMMA have Raman resonances. The method can be extended to multiple vibrational modes.     

Micro‐Raman Spectroscopy Study of Human Pterygium and Conjunctival Melanocytic Nevi

Pterygium, a common ophthalmic disease that is caused by fibrovascular growth of conjunctiva and conjunctival melanocytic nevi that is another conjunctival disease, are detected by Raman spectroscopy in the present study. We find that there is an obvious increase in the intensity at the peak of 1,583 cm^?1 that is assigned to C=C unsaturated fatty acids stretch of lipids in the pterygium tissue, and 1,639 cm^?1 also increased which belongs to amide I. Also, PCA (Principal Component Analysis) was used to classify the normal conjunctiva from the pterygium tissue. For the conjunctival melanocytic nevi, the intensity of Raman spectrum region between 1,550 cm^?1 and 1,650 cm^?1 that belong to protein has increased, which indicates that the content of protein in conjunctival melanocytic nevi is more richer than the normal ones. SCANNING 34: 395–398, 2012. © 2012 Wiley Periodicals, Inc.     

Biochemical fingerprints of human papillomavirus infection and cervical dysplasia using cervical fluids: Spectral pattern investigation

The Pap smear is the primary screening tool for invasive cervical cancer resulting from a persistent infection with oncogenic human papillomavirus (HPV); however, there are the problems such as the inability to distinguish between HPV infection and cervical dysplasia and a low sensitivity remain. We present preliminary findings of a label‐free method to detect and classify HPV infection and cervical dysplasia using human cervical fluids. Three experimental groups, defined as normal, HPV‐positive, and cervical dysplasia, were evaluated through their Raman spectral patterns for noise‐independence, high reproducibility, and uniformity. Clinical diagnosis was performed through liquid‐based cervical cytology, HPV test, and cervical histologic examination. Healthy cervical fluids showed a strong Raman intensity at 877 cm^?1 (symmetric C–C stretching), and at 963 cm^?1 (phosphate), compared to a reference Raman peak at 1003 cm^?1 (phenylalanine symmetric ring breath). The HPV‐positive cervical fluids showed a strong intensity of a Raman peak at 1448 cm^?1 corresponding to C–H deformation vibration mode and the highest similarity between the central and ring zones among the three groups. The cervical dysplasia fluids showed the presence of strong peaks compared to the control and HPV‐positive groups. In addition, different Raman spectra were acquired according to HPV type. Therefore, all ranges of cervical fluid‐induced Raman spectra could be used to detect the presence of cervical pre‐cancer. Raman peak‐gated assessment provides a label‐free and nondestructive tool for the clinical diagnosis of HPV infection and cervical precancerous changes.     

Mechanisms of radiation damage in beam‐sensitive specimens,for TEM accelerating voltages between 10 and 300 kV

Ionization damage (radiolysis) and knock‐on displacement are compared in terms of scattering cross section and stopping power, for thin organic specimens exposed to the electrons in a TEM. Based on stopping power, which includes secondary processes, radiolysis is found to be predominant for all incident energies (10–300 keV), even in materials containing hydrogen. For conducting inorganic specimens, knock‐on displacement is the only damage mechanism but an electron dose exceeding 1000 C cm^?2 is usually required. Ways of experimentally determining the damage mechanism (with a view to minimizing damage) are discussed. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

Accurate measure of laser irradiance threshold for near-infrared photo-oxidation with a modified confocal microscope

Femtosecond mode‐locked lasers are now being used routinely in multiphoton fluorescence and autofluorescence spectroscopy, are just beginning to be used in refractive surgery, and may be used in the future diagnosis of skin cancer. Pulses from these lasers induce non‐linear effects in resultant tissue interactions. Using a modified confocal microscope with dispersion compensation and accurate measurements of beam diameter, a very low threshold was measured for photochemical oxidation in cultured cells. The measured threshold showed non‐linear photo‐oxidation at a peak irradiance and photon‐flux density of 8.4 × 10⁸ W cm^?2 and 3.4 × 10²⁷ photons cm^?2 s^?1, respectively (90‐fs pulse). The impact of these findings is significant to those using ultrashort lasers because they provide a tangible reference point (microscope‐independent) for the generation of photo‐oxidative stress in laser‐exposed tissues, and because they highlight the importance of dispersion compensation in minimizing collateral tissue damage.     

Near-field and confocal surface-enhanced resonance Raman spectroscopy at cryogenic temperatures

For laser spectroscopy at variable temperatures with high spatial resolution a combined scanning near‐field optical and confocal microscope was developed. Rhodamine 6G (R6G) dye molecules dispersed on silver nano‐particles or nano‐clusters were investigated. For optical excitation of the molecules, either an aperture probe or a focused laser spot in confocal arrangement were employed. Raman spectra in the wavenumber range between 300 cm^?1 and 3000 cm^?1 at room temperatures down to 8.5 K were recorded. Many of the observed Raman lines can be associated with the structure of the adsorbed molecule. Intensity fluctuations in spectral sequences were observed down to 77 K and are indicative of single molecule sensitivity.     

Mapping the chemistry of resinite,funginite and associated vitrinite in coal with micro‐FTIR

Micro‐FTIR mapping is a powerful tool for nondestructive, in situ chemical characterization of coal macerals at high resolution. In this study, the chemistry of resinite, funginite and associated vitrinite is characterized via reflectance micro‐FTIR for Cenozoic high volatile C bituminous coals from Colombia. In comparison with the micro‐FTIR spectra of vitrinite and inertinite, the corresponding spectra of liptinite macerals in the same coals are characterized by stronger aliphatic CH_x absorbance at 3000–2800 and 1460–1450 cm^?1, but less intense aromatic C=C ring stretching vibration and aromatic CH_x out‐of‐plane deformation at 700–900 cm^?1. The aliphatic components in resinite have the longest carbon chains and are least branched, bestowing the highest hydrocarbon generation potential on resinite among the three macerals studied. In contrast, funginite exhibits the strongest aromatic character, the highest aromaticity, the lowest ‘A’ factor values and the lowest C=O/C=C ratios among the three maceral groups. Vitrinite generally displays intermediate chemical characteristics. Reflectance micro‐FTIR mapping of coal samples further confirms the aliphatic character of resinite and the aromatic nature of funginite. In addition, chemical mapping of resinite and adjacent vitrinite shows that vitrinite immediately adjacent to resinite displays higher aliphatic CH_x stretching intensity than more distant vitrinite, suggesting that chemical components from resinite can diffuse over short distances into adjacent vitrinite, specifically causing hydrogen enrichment. It needs to be pointed out, however, that the region of influence is localized and limited to a narrow zone, whose extent likely depends on resinite's properties, such as its size and aliphatic material content. This way, the chemical map of resinite and associated vitrinite provides direct evidence of the intermaceral effects occurring during the peat forming stage or during later coalification. No influence of funginite (primarily fungal spores and sclerotia) on the chemistry of adjacent vitrinite has been demonstrated, which is likely due to the highly aromatic structure of this type of funginite.     

Changes in nail keratin observed by Raman spectroscopy after Nd:YAG laser treatment

Lasers and photodynamic therapy have been considered a convergence treatment for onychomycosis, which is a fungal infection on the nail bed and nail plate. Laser therapies have shown satisfactory results without significant complications for onychomycosis; however, the mechanism of clearing remains unknown. In this work, we investigated changes in the chemical structure of nail keratin induced by Nd:YAG laser using Raman spectroscopy. Toe nails with onychomycosis were treated with 1064 nm Nd:YAG laser. After laser treatment, the disulfide band (490–590 cm^?1) of nail keratin was rarely observed or was reduced in intensity. The amide I band (1500–1700 cm^?1) also showed changes induced by the laser. The α‐helical (1652 cm^?1) structures dominated the β‐sheet (1673 cm^?1) in nontreated nail, but the opposite phenomenon was observed after laser treatment.     

Application of a high power Yb fiber‐based laser compatible with commercial optical parametric oscillator for coherent anti‐stokes raman scattering microscopy

Coherent anti‐Stokes Raman scattering (CARS) microscopy is a powerful tool for chemical analysis at a subcellular level, frequently used for imaging lipid dynamics in living cells. We report a high‐power picosecond fiber‐based laser and its application for optical parametric oscillator (OPO) pumping and CARS microscopy. This fiber‐based laser has been carefully characterized. It produces 5 ps pulses with 0.8 nm spectral width at a 1,030 nm wavelength with more than 10 W of average power at 80 MHz repetition rate; these spectral and temporal properties can be slightly modified. We then study the influence of these modifications on the spectral and temporal properties of the OPO. We find that the OPO system generates a weakly spectrally chirped signal beam constituted of 3 ps pulses with 0.4 nm spectral width tunable from 790 to 930 nm optimal for CARS imaging. The frequency doubling unconverted part is composed of 7–8 ps pulses with 0.75 nm spectral width compatible with CARS imaging. We also study the influence of the fiber laser properties on the CARS signal generated by distilled water. In agreement with theory, we find that shorter temporal pulses allow higher peak powers and thus higher CARS signal, if the spectral widths are less than 10 cm^?1. We demonstrate that this source is suitable for performing CARS imaging of living cells during several hours without photodamages. We finally demonstrate CARS imaging on more complex aquatic organisms called copepods (micro‐crustaceans), on which we distinguish morphological details and lipid reserves. Microsc. Res. Tech. 77:422–430, 2014. © 2014 Wiley Periodicals, Inc.     

Microinfrared reflection spectroscopic mapping: application to the detection of hydrogen-related species in natural quartz

A new method of microinfrared reflection spectroscopy and mapping analysis is briefly introduced. It was used to detect distributions and structures of hydrogen‐related species (e.g. H₂O, SiOH and SiH) in plastically deformed natural quartz. We used a Fourier transform‐infrared spectrometer with a microscopic imaging system fully automated for all microscope functions (e.g. focusing, aperture, stage motion and measurements). Mapping can be made in thin sections with a thickness of 50 µm at room temperature and low temperatures (77 K) using a liquid N₂ cooling system. Infrared reflection spectra were obtained by five scans for each point with a range from 4000 to 400 cm^?1. The spectra were measured five times within about 2.5 s at each position. The scanning interval was 100–150 µm using a 100 × 100 µm² aperture. All obtained spectral data were stored in computer memory to construct two‐dimensional mappings of infrared absorption. From the comparisons between infrared mapping images and deformation microstructures, in addition to the molecular H₂O around 3600–3400 cm^?1, the hydrogen‐related point defects (i.e. SiOH and SiH) around 970–900 cm^?1 within quartz grains and between grain boundaries increased with decreasing grain sizes (increasing plastic strain). The method can detect the SiOH and SiH along grain boundaries that enhance the hydrolytic weakening of natural quartz.     

3D‐quantum interferometer using silicon microring‐embedded gold grating circuit

A 3D (three‐dimensional) quantum interferometer consisting of a silicon microring circuit proposed. The interferometer based on the electron spin cloud projections generated by microring‐embedded gold grating. The electron cloud oscillations result from the excitation of the gold grating at the center of the silicon microring by the dark soliton pulse of 1.50 μm center wavelength. The electron cloud spin‐down, spin‐up automatically formed in the two axes (x, y, respectively) and propagated along the z‐axis. In this proposal, the sensing mechanism of the circuit is manipulated by varying the reflector gold lengths of the sensing arm. The electron cloud spin coupled and changed by changing the gold lengths. The sensitivity measurement of the 3D quantum interferometer for three gold layer lengths of 100 nm, 500 nm, and 1,000 nm is (47.62 nm fs^?1, ±0.4762 fs^?1, ±0.01 nm^?1), (238.10 nm fs^?1, ±0.4762 fs^?1, ±0.002 nm^?1), (476.20 nm fs^?1, ±0.4762 fs^?1, ±0.001 nm^?1), respectively. The used circuit parameters are the real ones that can be fabricated by the currently available technology. Moreover, the silicon micro ring circuit acts as a plasmonic antenna, which can apply for wireless quantum communication. The electron cloud spin projection space–time control can apply for quantum cellular automata.     

Quantification of anhydrous ethanol and detection of adulterants in commercial Brazilian gasoline by Raman spectroscopy

Raman spectroscopy can be used to evaluate the quality of fuels in a remote, rapid, and nondestructive manner without the need for reagents. In this study, Raman was used to quantify anhydrous ethanol in commercial gasoline and to detect peaks due to compounds commonly used for the adulteration of commercial gasoline. Samples of commercial gasoline were collected from fuel stations in the region of Santos, SP, Brazil. Samples of naphtha from the refinery, pure ethanol, and ethanol diluted in distilled water at concentrations close to the range used in the gasoline were also obtained and characterized. Raman spectra were collected using a dispersive Raman spectrometer (830?nm, 2?cm^?1 resolution in the 400–1800?cm^?1 spectral range). As expected, the spectra of commercial gasoline showed pronounced peaks of naphtha and ethanol. By using the peak intensities of the ethanol diluted in water, the ethanol concentration was found to be in the range of 27%?±?1% in most of the samples; some samples presented ethanol concentrations as high as 28.8%, suggesting adulteration. Some samples presented peaks at 766, 798, and 995?cm^?1 with higher intensities, suggesting the presence of an adulterant with organic characteristics, such as solvents with aromatic rings. Raman spectroscopy has been shown to be effective in determining the adulteration of commercial gasoline, which may contribute to rapid quality control of fuels at the point of sale.     

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.     

Characterization of an ultraviolet irradiation chamber to monitor molecular photodegradation by Raman spectroscopy

A homemade ultraviolet chamber is reported to induce photochemical changes with characterization by Raman spectroscopy. The equipment has compartments for ultraviolet-A (UV-A) (8 lamps of 8 W) and for UV-A + ultraviolet-B (UV-B) irradiation (4 lamps of 26 W and 1 lamp of 15 W). The irradiance was measured 3, 5, and 10 cm from the light sources. The maximum irradiance was obtained at 3 cm (UV-A: 2.66 mW/cm² and UV-A + UV-B: 4.30 mW/cm²). The chamber internal temperature was stabilized at 30°C after 1 hr of operation with an internal relative humidity of approximately 45%. 10% Collagen was irradiated with UV-A at 2.0 mW/cm² for 3 hr with changes in Raman peaks at 1253, 1271, 1453, and 1660 cm^?1 indicating changes in conformation. 5% Atenolol was irradiated with UV-A + UV-B at 4.30 mW/cm² for 8 hours with changes to Raman peaks at 822, 1186, 1206, 1248, and 1618 cm^?1. A commercial insect repellent was irradiated with UV-A + UV-B at 4.30 mW/cm² for 8 hr and decreases in Raman intensity were observed at 526, 690, 1003, and 1606 cm^?1 due to degradation of N, N-diethyl-m-toluamide. The results demonstrate proper operation of the irradiation chamber with Raman spectroscopic monitoring.     

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.     

USE OF DISPERSIVE RAMAN SPECTROSCOPY IN THE DETERMINATION OF UNSATURATED FAT IN COMMERCIAL EDIBLE OIL- AND FAT-CONTAINING INDUSTRIALIZED FOODS

The present study aims at the use of Raman spectroscopy in the quantification of unsaturated fats in fat-containing foods, compared to the information available in the Nutritional Table, to obtain a non-destructive optical quantification of unsaturation. Raman spectra of edible oil, margarine, mayonnaise, hydrogenated fat, and butter were obtained with a near-infrared Raman spectrometer (830 nm). By analyzing selected bands in the regions of 1750, 1660, 1440, 1300, and 1260 cm^?1, the amount of total and unsaturated fat of samples of oil, margarine, and mayonnaise were correlated with the information displayed in the Nutritional Table. The amount of unsaturated trans fat in selected samples was correlated to the Raman shift of 1660 cm^?1. Dispersive Raman spectroscopy was shown to be effective in quantifying the unsaturated fats in oil, margarine, and mayonnaise, and trans fat in hydrogenated oils and butter.     

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.