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Laser-stimulated fluorescence (LSF) imaging is a versatile observational technique that has a multitude of paleontological applications. Both automated and manual systems can be used to scan or otherwise observe fossils under laser illumination. A series of common steps apply to any LSF work, these are detailed below.
Laser light is concentrated on a specimen either as a point source for microscopic work, as a divergent light cone for smaller-sized specimens (with the aid of a laser diffuser), or a collimated beam (in which all light rays are parallel) is raster scanned over very large specimens. Since the laser is very bright, it must be blocked with an appropriate filter that still allows the longer wave fluorescence signal to pass through. Proper precautions using laser-blocking protective glasses and manufacturer’s safety protocol should be followed.
The equipment used with this methodology depends on the exact wavelength of light produced by the laser. Specialized light-blocking longpass filters, often used in astronomy, are best-suited for these methods. These particular filters will allow all wavelengths of light longer than a certain wavelength to pass through the filter, however, it will stop all shorter wavelengths. For instance, a red-orange longpass filter (LP580, MidOpt) will allow 91–95% of light between the wavelengths of 600–1100 nm, however, the transmission sharply decreases between 600–520 nm, and by 510 nm, no light passes through the filter (www.midopt.com). A 477 nm blue laser would be efficiently blocked by this filter, but will still allow imaging of longer fluorescent wavelengths. The laser wavelengths and filters for each particular specimen were chosen experimentally via the trial and error method, a procedure that we believe is reasonable given the simplicity of this technique. The setup parameters for each of the five case histories presented in this study are given in Table 1.
Standard UV bulbs can be used in addition to lasers in order to cover a broad range of the light spectrum. Imaging is done in both UVA from 315–400 nm and UVB from 280–315 nm. When working with UV light, photographs can be taken both with and without filters due to the low UV sensitivity of digital camera CCD (charge-coupled device) chips.
No special digital cameras are needed to photograph specimens using laser fluorescence. Typically, digital single lens reflex cameras (DSLRs) capable of manual time exposures (e.g. Nikon D610) with either wide angle or macro lenses are sufficient. Ideally, the photography should be done in a darkroom, basement, or office without windows or with blackout curtains, as any influence of natural light will reduce the clarity of the fluorescence. The use of a tripod is necessary, as the exposure time during photography is typically long—up to several minutes, although this may not be the case for close macro photography. The aperture setting (f-value) should be as low as possible for long-exposure shots.
Multiple types of laser light sources can be used. The more powerful the laser, the better and brighter the fluorescence. For the experiments outlined here, class III lasers in the 300–500 mW category were used. These were well below the threshold that results in radiation damage to the specimens studied. A lab laser, which plugs into the wall and is fairly static, and a high-powered laser pointer that runs off of CR123A lithium batteries, have both been used successfully depending on the locality of the specimen. The benefit of using a lab laser is that it can be used for hours at a time without overheating. It is typically used for precision work and photographing larger specimens. A high-power laser pointer is more portable and adjustable than a lab laser, however it can only be used for ~5 minutes, or else it will overheat and become damaged. If the photographer knows what f-value and shutter speeds are necessary for photography, a laser pointer can be used to great effect. It is excellent for macro photography in the field due to its portability.
A laboratory setup for table-top-sized specimens would typically hold the laser on a fixed mount (Fig 1). The laser itself emits a collimated beam, which results in only a small dot of illumination. This beam can be used as is for maximum flux or be expanded using a diffuser (ARF used a 20-degree diffraction diffuser from Thorlabs). The smaller the angle of the diffuser, the better—i.e. 20 degrees would be better than 50 degrees, as it restricts the beam to a narrower angle and results in a brighter and smaller area of illumination, even if the laser is placed further away from the specimen. The laser should illuminate as much of the specimen as possible, and the diffuser’s cone angle changes the area covered by the laser depending on the laser-to-specimen distance.
Larger specimens can be scanned using a custom device (Fig 2A and 2B). A Powel laser line lens projects a laser line in the Y direction that evenly distributes the laser energy over the length of the line (Fig 2C). A motor scans the entire assembly in the X direction (Fig 2A and 2B). This allows specimens of almost any size to be imaged. The exposure time of the DSLR camera should cover one or more of the X direction scans of the specimen.
For a microscope setup, the collimated laser beam is directed through one of the illumination ports or projected directly onto the specimen. The emitted light, laser and fluorescence, comes back through the microscope’s optical train where a longpass filter is placed either before the objective lens or internally in a filter slot to block the intense laser light. The fluorescence can then be observed and photographed in detail.
Specimen sources for each case history:
Case history 1: Burke Museum of Natural History and Culture, UWBM 103073—feather from Green River Fm.; UWBM 103074—feather from Parachute Member of Green River Fm.
Case history 2: Department of Land and Resources of Liaoning Province, LVH 0026—fish specimen from Jiufotang Fm. [20 (link)]
Case history 3: UWBM 103075—microfossils from Brule Fm.; UWBM 103076—microfossils from Hell Creek Fm.
Case history 4: Gobero specimen housed in the University of Chicago Research Collection, G1B2—juvenile female skeleton from mid-Holocene lake deposits
Case history 5: Institute of Vertebrate Paleontology and Paleoanthropology, Beijing, China, IVPP V13320—Microraptor skull from Jiufotang Fm. [21 (link)]

Chemical methods: Commercially available compounds were used without further purification. All fluorogenic substrates for the labeling of SNAP-tag fusion proteins were prepared by reacting the building block CBG-NH₂ (New England Biolabs) with commercially available N-hydroxysuccinimide esters of the corresponding fluorophores and amines of the corresponding quenchers. ATTO-488 NHS was purchased from ATTO-TEC GmbH (Siegen, Germany). Tide Fluor 3 (TF3) NHS, Tide Fluor 5 (TF5) NHS, Tide Quencher 2 (TQ2) acid, Tide Quencher 3 (TQ3) acid were purchased from AAT Bioquest, Inc. (Sunnyvale, CA). Dabcyl C2 amine and QXL670 C2 amine were purchased from AnaSpec, Inc. (Fremont, CA). DY-549 NHS and DY-647 NHS were purchased from Dyomics GmbH (Jena, Germany). Alexa Fluor 647 NHS, QSY-7 amine, and QSY-21 NHS were purchased from Life Technologies Co. (Carlsbad, CA). IRDye QC-1 NHS was provided by LI-COR Biosciences (Lincoln, NE). QSY-21 amine, TQ2 amine, TQ3 amine, and IRDye QC-1 amine were synthesized by reacting N-Fmoc-1,2-diaminoethane hydrobromide (Sigma–Aldrich) with commercially available QSY-21 NHS, TQ2 acid, TQ3 acid, and IRDye QC-1 NHS, respectively. Due to the confidential or proprietary nature of the majority of fluorophores and quenchers used in this study, very limited information about chemical structures is available from dye manufacturers.
Purification and analysis of substrates: Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed on an Agilent LCMS Single Quad System 1200 Series (analytical) and Agilent 1100 Preparative-scale Purification System (semi-preparative). Analytical HPLC was performed on a Waters Atlantis T3 C18 column (2.1×150 mm, 5 μm particle size) at a flow rate of 0.5 mL min⁻¹ with a binary gradient from solvent A (0.1 % aq. formic acid) to solvent B (acetonitrile with 0.1 % formic acid) and monitored by UV–visible absorbance at 280 nm and at the absorption maximum of each fluorophore. Semi-preparative HPLC was performed on a VYDAC 218TP series C18 polymeric reversed-phase column (22×250 mm, 10 μm particle size) at a flow rate of 20 mL min⁻¹ using a water/acetonitrile gradient with trifluoroacetic acid (0.1 %) or 1 m triethyl ammonium bicarbonate buffer (0.1 %). Mass spectra were recorded by electrospray ionization (ESI) on Agilent 6210 Time-of-Flight (TOF) LC/MS System. UV spectra were recorded on a Beckman DU 640B Spectrophotometer.
Synthesis of fluorogenic substrates: Reactions (1–2 μmol scale) were performed at room temperature in N,N-dimethylformamide in the presence of CBG-NH₂ (1.0 equiv), triethylamine (2.0 equiv), and the fluorophore N-hydroxysuccinimidyl ester (1.0 equiv). The mixture was stirred for 12 h. Then the corresponding quencher amine (1.1 equiv), HBTU (1.5 equiv), and triethylamine (2.0 equiv) were added. The reaction completion was monitored by LCMS. Typically, after 1 h stirring, the mixture was concentrated, purified by RP-HPLC and lyophilized. Each substrate was analyzed by high-resolution mass spectrometry and UV absorption. Isolated yields are given in parentheses and are not optimized. The following substrates were purified using a water/acetonitrile gradient: SNAP-Surface 488 (70 %): ESI-TOFMS m/z 842.2027 [M+H]⁺ (calcd for C₃₈H₃₅N₉O₁₀S₂, m/z 842.2021); UV (pH 7.5) λ_max=507 nm. CBG-488-DABCYL (32 %): ESI-TOFMS m/z 1207.3854 [M+H]⁺ (calcd for C₅₈H₅₈N₁₄O₁₂S₂, m/z 1207.3873); UV (pH 7.5) λ_max=505 nm. CBG-488-TQ2 (28 %): ESI-TOFMS m/z 1320.4156 [M+H]⁺ (calcd for C₆₃H₆₅N₁₅O₁₂S₃, m/z 1320.4172); UV (pH 7.5) λ_max=503 nm. SNAP-Surface 549 (76 %): ESI-TOFMS m/z 1069.2551 [M−H]⁻ (calcd for C₄₆H₅₄N₈O₁₄S₄, m/z 1069.2570); UV (H₂O) λ_max=555 nm. CBG-TF3 (51 %): ESI-TOFMS m/z 783.3248 [M+H]⁺ (calcd for C₄₃H₄₂N₈O₇, m/z 783.3249); UV (MeOH) λ_max=545 nm. CBG-TF3-DABCYL (21 %): ESI-TOFMS m/z 1076.4851 [M+H]⁺ (calcd for C₆₀H₆₁N₁₃O₇, m/z 1076.4890); UV (MeOH) λ_max=556 nm. SNAP-Surface 647 (68 %): ESI-TOFMS m/z 895.3237 [M+H]⁺ (calcd for C₄₅H₅₀N₈O₈S₂, m/z 895.3266); UV (EtOH) λ_max=652 nm. CBG-TF5 (63 %): ESI-TOFMS m/z 1203.2896 [M+H]⁺ (calcd for C₅₄H₅₈N₈O₁₆S₄, m/z 1203.2926); UV (MeOH) λ_max=655 nm. The following substrates were purified using a water/acetonitrile gradient with trifluoroacetic acid (0.1 %): CBG-549-TQ3 (13 %): ESI-TOFMS m/z 808.7301 [M−2H]²⁻ (calcd for C₇₃H₈₅N₁₅O₁₈S₅, m/z 808.7328); UV (MeOH) λ_max=558 nm. CBG-549-QSY7 (54 %): ESI-TOFMS m/z 933.8112 [M+2H]²⁺ (calcd for C₉₃H₁₀₃N₁₃O₁₉S₅, m/z 933.8127); UV (MeOH) λ_max=559 nm. CBG-TF3-TQ3 (17 %): ESI-TOFMS m/z 1260.5157 [M+H]⁺ (calcd for C₆₇H₆₉N₁₅O₉S, m/z 1260.5196); UV (MeOH) λ_max=556 nm. SNAP-Surface Alexa Fluor 647 (87 %): ESI-TOFMS m/z 1111.2993 [M+H]⁺ (calcd for C₄₉H₅₈N₈O₁₄S₄, m/z 1111.3028); UV (MeOH) λ_max=651 nm. CBG-TF5-QXL670 (47 %): ESI-TOFMS m/z 925.8117 [M+2H]²⁺; UV (MeOH) λ_max=657 nm. CBG-TF5-QSY21 (67 %): ESI-TOFMS m/z 954.7859 [M+2H]²⁺ (calcd for C₉₇H₉₈N₁₃O₁₉S₅, m/z 954.7887); UV (MeOH) λ_max=656 nm. The following substrates were purified using water/acetonitrile gradient with 1 m triethylammonium bicarbonate buffer (0.1 %): CBG-549-QC1 (11 %): ESI-TOFMS m/z 1121.7978 [M+2H]²⁻ (calcd for C₁₀₁H₁₂₅ClN₁₂O₂₈S₈, m/z 1121.8057); UV (EtOH) λ_max=561 nm. CBG-AF647-QC1 (22 %): ESI-TOFMS m/z 1141.8104 [M−2H]²⁻ (calcd for C₁₀₃H₁₂₈ClN₁₃O₂₈S₈, m/z 1141.8150); UV (MeOH) λ_max=651 nm. CBG-647-QC1 (31 %): ESI-TOFMS m/z 1035.3349 [M+H]²⁺ (calcd for C₉₉H₁₂₀ClN₁₃O₂₂S₆, m/z 1035.3376); UV (EtOH) λ_max=653 nm. Detailed experimental protocol and ¹H NMR spectrum for CBG-549-QSY7 (Scheme S1) can be found in the Supporting Information. Substrates were further characterized by ESI-TOF mass spectrometry after their binding to the SNAP_f protein (Table S2).
Expression constructs (Figure S2): pSNAP_f was constructed by insertion of the cDNA encoding SNAP_f, synthesized by IDT, between the restriction sites EcoRI and SbfI of pSNAP-tag(m) (New England Biolabs). This SNAP-tag variant, SNAP_f, contains 19 amino acid substitutions and an additional 24-residue deletion at the C-terminus compared to the wild-type AGT. Constitutive expression of the SNAP_f is under the control of a CMV promoter. The cDNA encoding the CLIP_f was introduced between the EcoRI and SbfI sites of pSNAP_f, resulting in pCLIP_f. pSNAP_f-tag(T7) and pCLIP_f-tag(T7) were constructed by replacing the SNAP-26 m coding region of pSNAP-tag(T7)-2 using the unique EcoRI and SbfI sites with the coding regions of SNAP_f and CLIP_f, respectively.
The mouse EGF coding sequence was fused in-frame to the 5′-end of SNAP_f and CLIP_f, and a hexahistidine tag (His₆) was fused to the 3′-end of SNAP_f and CLIP_f in pSNAP_f-tag(T7) and pCLIP_f-tag(T7), respectively. The resulting plasmids pEGF-SNAP_f-His₆ and pEGF-CLIP_f-His₆ were used for expression of EGF-SNAP_f and EGF-CLIP_f fusion proteins in E. coli and subsequent affinity purification by Ni-NTA agarose (Qiagen). A linker encoding the signal sequence of EGFR, formed by annealing 5′-CTAGC ATGCG ACCCT CCGGG ACGGC CGGGG CAGCG CTCCT GGCGC TGCTG GCTGC GCTCT GCCCG GCGAG TCGGG CTG-3′- and 5′-AATTC AGCCC GACTC GCCGG GCAGA GCGCA GCCAG CAGCG CCAGG AGCGC TGCCC CGGCC GTCCC GGAGG GTCGC ATG-3′, was inserted into the 5′-MCS of the pSNAP_f vector using the unique NheI and EcoRI sites (underlined). Subsequently the coding sequence of mature EGFR (GeneCopoeia) was amplified by PCR and subcloned into the plasmid described above using the unique SbfI and NotI sites, creating pSNAP_f-EGFR. SNAP_f-β-tubulin was generated from the human β-tubulin coding sequence (Open Biosystems) which was amplified by PCR and fused in-frame to the 5′-end of SNAP_f in the pSNAP_f vector.
Fluorescence in-gel detection: SNAP_f protein was labeled at 37 °C for 30 min in the presence of SNAP_f (5 μm), BG conjugate (10 μm) and DTT (1 mm) in PBS. The samples were submitted to electrophoresis on a 10–20 % Tris-glycine gel under denaturing conditions. The gels were scanned using a Typhoon 9400 imager at 300 V PMT with a 488/526 nm (Figure 2 A, 488 in green), 532/580 nm (Figure 2 B, 549 in orange) or 633/670 nm excitation/emission filter set (Figure 2 C, TF5 and Alexa Fluor 647 in red).
Assay of quenching efficiency: Fluorescence signals of the SNAP_f proteins labeled with a fluorophore from a quenched or non-quenched substrate were analyzed with a FLEXstation scanning fluorometer (Molecular Devices). The reactions were performed in 96-well plates (Costar) and the fluorescence was measured at the appropriate wavelength. Reactions were carried out with dye (5 μm) and DTT (1 mm) in PBS in the presence or absence of SNAP_f protein (10 μm). SNAP-Surface 488 and its fluorogenic derivatives were excited at 488 nm and measured at the maximum emission wavelength of 526 nm. SNAP-Surface 549, CBG-TF3 and their fluorogenic derivatives were excited at 546 nm and measured at the maximum emission wavelength of 580 nm. Fluorescence of SNAP-Surface 647, SNAP-Alexa Fluor 647, CBG-TF5 and their fluorogenic derivatives was read at 636 nm with maximum emission of 670 nm. Fluorescence was followed in 5 min intervals over 2 h at 25 °C. Quenching efficiencies were calculated by the equation E=1−(I_FD/I_SNAPf), where I_FD indicates fluorescence intensity of free dyes and I_SNAPf indicates fluorescence intensity of labeled SNAP_f protein at the end of the 2 h reaction.
Kinetic study: Labeling reactions were carried out at 22 °C in the presence of dye (5 μm), SNAP_f protein (1 μm) and DTT (1 mm) in PBS. At each of the following time points: 0, 15, 30 or 45 s, 1, 2, 4, 8, 16, 32 or 64 min, 18 μL of the labeling reaction was removed and added to a microfuge tube containing 18 μL of 3×Red SDS-PAGE loading buffer (New England Biolabs). After boiling the samples for 5 min, each sample (7.5 μL) was loaded on a 10–20 % Tris-glycine gel (Invitrogen). Following separation of proteins and free dyes on SDS-PAGE, the labeled SNAP_f protein was detected with fluorescence imager Typhoon 9400 (GE Healthcare). Gel scanning was performed with appropriate filter sets: excitation at 488 nm and emission at 526 nm for SNAP-Surface 488 and its fluorogenic derivatives; excitation at 533 nm and emission at 580 nm for SNAP-Surface 549, CBG-TF3 and their fluorogenic derivatives; excitation at 633 nm and emission at 670 nm for SNAP-Surface 647, SNAP-Alexa Fluor 647, CBG-TF5 and their fluorogenic derivatives. The imaging data were quantified with ImageQuant TL software (GE Healthcare). The data were fitted to an exponential rise model using the KaleidaGraph 4.0 software (Synergy Software) to get the pseudo-first-order rate constants. Second-order rate constants were then obtained by dividing the pseudo first-order constant by the concentration of substrate.
Quantification of SNAP_f-β-tubulin in cell lysates: To generate a standard curve of fluorescence intensity versus SNAP_f protein concentration, purified SNAP_f protein (25 μL) at a final concentration of 0.025, 0.05, 0.075, 0.1, 0.125, and 0.25 μm were incubated with CBG-488-TQ2 (2 μm, 25 μL, final concentration 0.5 μm) and of cell lysate (50 μL) from nontransfected U2OS cells at room temperature for 4.5 h. The reaction was performed in triplicate in a 96-well plate (Costar). The fluorescence intensity was recorded at 526 nm emission maximum upon excitation at 488 nm and plotted against SNAP_f protein concentration. The curve was fitted to a linear equation.
The concentration of SNAP_f-β-tubulin was measured from cell lysates of U2OS cells stably expressing SNAP_f-β-tubulin. Cells grown at 37 °C in phenol red-free DMEM medium supplemented with 10 % fetal bovine serum (FBS), L-glutamine (2 mm), penicillin (100 units per mL), streptomycin (100 μg mL⁻¹) and G418 (200 μg mL⁻¹) were harvested from a 75 cm²; cell culture flask (BD Falcon) with 0.25 % trypsin treatment, then washed and spun down. The cell pellet was lysed in 500 μL of CelLytic M cell lysis reagent (Sigma–Aldrich) for 15 min at room temperature. Total protein concentration was determined by the Bradford assay. The cell lysate was serially diluted with PBS buffer (1:1, 1:2, 1:4, 1:8, and 1:16) to generate cell lysate samples with various total protein concentrations. 50 μL of each dilution was mixed with 1 μm CBG-488-TQ2 (50 μL, final concentration 0.5 μm) and incubated at room temperature for 4.5 h. The reaction was performed in triplicate in a 96-well plate and the fluorescence intensity was recorded at 526 nm upon excitation at 488 nm. The fluorescence intensities were converted to SNAP_f-β-tubulin protein concentrations by using the standard curve generated for SNAP_f. The total protein concentration (mg mL⁻¹) was plotted against the concentration of SNAP_f-β-tubulin in the cell lysate (μm). The signal-to-noise (S/N) ratios were determined as S/N=(I_F−I_B)/SD, where I_F is the average fluorescence intensity, I_B is the average background intensity, and SD is the standard deviation of background. The signal-to-background (S/B) ratios were determined as S/B=I_FT/I_FNT, where I_FT is the average fluorescence intensity of transfected U2OS cells and I_FNT is the average fluorescence intensity of nontransfected U2OS cells.
Live cell labeling and imaging: Human embryonic kidney (HEK 293) cells stably transfected with pSNAP_f-EGFR were maintained at 37 °C in phenol red-free DMEM medium supplemented with 10 % fetal bovine serum (FBS), penicillin (100 units per mL), streptomycin (100 μg mL⁻¹) and G418 (200 μg mL⁻¹). Cells were seeded in Lab Tek II chambered coverglasses (Nalge Nunc Int). At 24 h post-seeding, cell membrane-localized SNAP_f-EGFR was labeled by incubation of live HEK 293 cells stably expressing SNAP_f-EGFR with SNAP-tag substrate (1 μm) for 30 min at 37 °C. Then SNAP-Surface Block (New England Biolabs) was added to the cells (final concentration 20 μm) to inhibit further labeling. Images were taken on a wide-field Axiovert 200 m Zeiss microscope using a 63× objective and fixed exposure setting. Cell nuclei were counterstained with Hoechst 33342. For imaging with medium removal, labeling was carried out as above, except that labeling medium was replaced with complete growth medium containing SNAP-Surface Block (20 μm). Images were processed using AxioVision 4.7 software.
EGF-CLIP_f isolation and labeling: Expression of recombinant EGF-CLIP_f-His₆ was performed in SHuffle T7 E. coli (New England Biolabs). EGF-CLIP_f-His₆ was purified from E. coli cell lysate using Ni-NTA Agarose (Qiagen). Analysis of protein expression and purification was done with Coomassie Blue-stained SDS-PAGE. Labeling of EGF-CLIP_f-His₆ was carried out with EGF-CLIP_f-His₆ (40 μm), CLIP-Surface 488 (15 μm) and DTT (1 mm) in PBS on ice for 4 h.
Colocalization of SNAP_f-EGFR and EGF-CLIP_f: HEK293 cells stably expressing SNAP_f-EGFR were labeled with 5 μm CBG-549-QSY7 (red) at 25 °C for 5 min. Cells were then incubated for 2 min with EGF-CLIP_f labeled with CLIP-Surface 488 (green) at 500 ng mL⁻¹ prior to imaging by confocal fluorescence microscopy. Cells were counterstained with Hoechst 33342 for nucleus (blue). Images were acquired on a Zeiss LSM 510 laser scanning confocal microscope using a 63X objective. Images were processed using LSM 510 Meta software.

Dual colour interphase FISH was performed on FFPE sections using commercial probe sets. For the evaluation of a potential chromosome 1q gain, a spectrum green labeled probe was used for test locus 1q25.3 and a spectrum orange labelled probe for control locus 1p36.31 (ZytoLight). For the evaluation of a potential chromosome 6q loss, a spectrum green labelled probe was used for test locus ESR1 at 6q25.1 and a spectrum orange labelled probe for the alpha satellite centromeric region of chromosome 6 (D6Z1) (ZytoLight). Signals were scored under a fluorescence microscope in 100 non-overlapping, intact nuclei under oil-immersion. If a cell showed three or more signals of the 1q test probe or the ratio of test probe-control probe inside one nucleus was more than one, signals were scored as chromosome 1q gain. If a cell showed one or less signals of the 6q test probe and/or the ratio of test probe-control probe inside one nucleus was ≤ 0.5, signals were scored as chromosome 6q loss. Tumor signals were scored as gains or losses for the whole area when at least 10% of the cells showed the gain or loss, as previously described [22 (link), 28 (link)].