NCI Optical Microscopy Laboratory

Software & Techniques

Delta Vision Elite

• Capabilities: This microscope is suited for live cell imaging (4D) and fixed cells (3D) imaging. This microscope produces high-quality wide-field images that may be improved by deconvolution. This system also has lasers that may be used for FRAP or photoactivation.
• Microscope: Applied Precision/GE Healthcare, Issaquah WA; consists of customized Olympus IX71 inverted microscope with Applied Precision proprietary optical sectioning stage (Long Travel 25 x 50 mm Flexure Stage); with live speed option – enables multi-color imaging without filter switches, with Ultimate focus – corrects for axial drift in 4D live time-lapse experiments, and with precision photokinetics optics components – generates a focused laser spot to target region of interest. The microscope is controlled bu SoftWoRx 6 software installed on a Linux-based workstation.
• Objectives: Olympus 100 X NA 1.4 WD 0.12 mm, 60X NA 1.4 WD 0.15 mm, 40X NA 0.6 WD 2.7-4.0 mm, 20X 0.45 NA WD 6.6-7.8 mm, 10X 0.4NA WD 3.1 mm.
• Filters: Excitation Wavelengths: DAPI = 390/18, CFP = 438/24, GFP/FITC = 475/28, YFP = 513/17, TRITC = 542/27, mCherry/Alexa594 = 575/25, Cy5 = 632/22; Single Band Pass Emission Filters: DAPI = 435/48, CFP = 475/24, GFP/FITC = 525/48, YFP = 548/22, TRITC = 597/45, mCherry/Alexa594 = 625/45, Cy5 = 679/34 Polychroic Beam Splitter suitable for DAPI, FITC, RD-TR-PE, CY5 Polychroic Beam Splitter suitable for DAPI, FITC, Alexa 594, CY5 and GFP/mCherry Polychroic Beam Splitter suitable for CFP/YFP/mCherry Dual bandpass EM filter for GFP/mCherry and triple bandpass filter for CFP/YFP/mCHerry fot multicolor imaging withought filter switching.
• Camera: Scientific CMOS camera, chip size 2560 x 2560 pixels, pixel size 6.6 x 6.5 um; 15-bit dynamic range, 54% quantum efficiency at the peak of 540 nm.
• Light Source: LED transmitted light module with 7-color combined InsightSSI, or 405 nm, 488 nm, 561 nm lasers.
• Accessories: a Clear environmental chamber with temperature (+7C to 45C) and CO2 control.

ELYRA Super-Resolution Microscope

• Capabilities: ELYRA PS.1 is an SR microscope system for Photoactivated Localization Microscopy (3D PALM) and Structured Illumination Microscopy (SIM), in other words, two instruments in one. The depth of focus for 3D PALM based on double-phase ramp is 1.4 um in three dimensions. Expected resolution in XYZ for PALM is / / 60 nm. Expected resolution for SIM is 70/70/150 nm. The regular confocal microscopes have an XYZ resolution of.
• Microscope: inverted microscope AxioObserver Z1 (Carl Zeiss, Inc, Thornwood, NY). Data collection is controlled by a Windows 7-based ZEN software.
• Objectives: Plan Apochromat 63X/1.4 NA Oil, Alpha Plan Apochromat 100X/1.46 NA Oil.
• Light Detectors: Andor 895 EM CCD for PALM and Pco.edge sCMOS for SIM.
• Lasers: 200 mW 488 nm, 200 mW 561 nm, 150 mW 640 nm
• Special features and accessories: Double-phase ramp for 3D PALM, Definite focus, 30C Environmental chamber.

Carl Zeiss LSM780 Microscope

Capabilities: Carl Zeiss LSM780 with 34 spectral detectors including high-sensitive GaASP is a point scanning confocal microscope that generates optical sections within 3D specimens. It can collect images in five colors (blue, green, orange, red and far red) and it is especially well suited for multicolor imaging, FRET, DNA damage experiments, photoactivation with 405 nm laser line, and biophysical assays such as FRAP and Number and Brightness.
• Microscope: inverted microscope AxioObserver Z1 (Carl Zeiss, Inc, Thornwood, NY). Data collection is controlled by a Windows 7-based ZEN software.
• Objectives: Alpha Plan Apochromat 100X/1.46 NA Oil, Alpha Plan Apochromat 63X/1.46 NA Oil, Plan Apochromat 40X/1.4 Oil, C-apochromat 40X/ 1.2 NA Water, LCI Plan Neofluar 25X/0.8 NA Water/Glycerol/Oil.
• Light Detectors: GaASP (32 channels), two PMT, a transmitted light detector
• Lasers: 60 mW 355 nm; 30 mW 405 nm, 25 mW Argon 458, 488, 514 nm; 20 mW 561 nm; 2 mW 594 nm; 5 mW 633 nm.
• Special features and accessories: Definite focus, CO2/heating control stage insert.

Custom-Built Single Molecule Tracking (SMT) Microscope

• Capabilities: This microscope is suited for tracking of single molecules in two color channnel using a third channel for a counterstain.. It is a wide-field microscope with Highly Inclined Laminated Optical Sheet (HILO) illumination, substantially reducing out-of-focus light and increasing signal to noise ratio. This system also may be used for the fast live imaging. Z-sectioning is currently not available on this system.
• Microscope: Custom-built system is based on Rapid Automated Modular Microscope System (RAMM, Applied Scientific Instrumentation, Eugene, OR) All lasers and cameras are synchronized using a microcontroller board (Arduino UNO), and images are collected using the open source microscope control software, Micro-Manager Open Source Microscopy Software (Open Imaging, Inc, San Francisco, CA).
• Objective: 100x 1.49 NA oil immersion objective lens (Olympus Scientific Solutions, MA, USA) The combination of objective lens and tube lens results in a total magnification of 111x, corresponding to a XY pixel size of 117 nm.
• Filters: Fluorescence emission is separated from scattered laser light by use of a quad-band dichroic (ZT405/488/561/647, Chroma Technology Corp., VT, USA), and the emission bands are separated by two long-pass filters (T588lpxr, T660lpxr), and emission filters (525/50, 609/58 and 736/128, Semrock, Inc., NY, USA) before being focused on three separate EMCCD cameras.
• Cameras: Three iXon Ultra 888, (Andor Technology Ltd., UK), with 200 mm tube lenses.
• Light source: The system has four laser lines: 405 nm (Stradus 405-100, Vortran Laser Technology, Sacramento, CA), 488 nm (OBIS 488 LS, Coherent, Inc., Santa Clara, CA), 561 nm (OBIS 561 LS, Coherent, Inc., Santa Clara, CA), and 647 nm (OBIS 647 LX, Coherent, Inc., Santa Clara, CA).
• Accessories: OKO lab stage insert with temperature and CO2 control.

Airyscan Carl Zeiss LSM880 Microscope

• Capabilities: Airyscan Zeiss LSM880 confocal microscope is capable of the conventional confocal imaging. In addition, it is equipped with Airyscan module. This module improves resolution in XYZ beyond the diffraction limit for the live cultures and small animals (zebrafish).
• Microscope: The Zeiss LSM880 confocal inverted microscope. Data collection is controlled by a Windows 10-based ZEN software.
• Objective: PlanApo 10X/0.45 NA; LD LCI P-APO 25X/0.8 NA; C-Apochromat 40X/ 1.2 NA Water; PlanApo 63X/1.4 NA; LD LCI P-APO 63X/1.2 NA Oil/Glyc; Alpha P-Apo 100X/ 1.46 NA
• Light detectors: GaASP detectors (3) and Airyscan detector.
• Lasers: Laser lines available are 405, 458, 488, 514, 561, 594, 633 and 730 nm.
• Special features and accessories: Definite focus, CO2/heating control stage insert, environmental chamber for live imaging.

3I Lattice Light Sheet Microscope

• Capabilities: Primary purpose – live imaging of cell cultures and thin tissue sections (≤ 100 um). Lattice Light Sheet technique offers fast simultaneous two-color imaging in 3D and 4D producing large volumes of high-resolution fluorescence imaging data, with low photo-toxicity. Lattice-generated light sheet illuminates only a thin slice of the cell compatible by thickness with a working distance of the objective. Thus, out-of-focus light and photobleaching of the probes are substantially reduced. In comparison to other imaging methods, the lattice light sheet technique (1) substantially increases a signal to noise ratio and reduces the light exposure, (2) allows ultra-thin optical sectioning (3) ensures extremely low phototoxicity, (4) substantially increases a signal to noise ratio, (5) allows very fast time lapse imaging on the scale of milliseconds (6) increases resolution (XYZ = 230 nm x 230 nm x 370 nm).
• Making a lattice light sheet: The innovative Lattice Light Sheet combines the flexibility of a Gaussian light sheet with the thin optical sectioning of a Bessel beam to achieve the best optical sectioning of any light sheet microscope. Cylindrical lenses stretch and collimate the beam to form a sheet projected onto a spatial light modulator (SLM). SLM generates an optical lattice of Bessel beams. Annular mask lengths the sheet. The sheet is dithered in X and swept in Z by galvo mirrors. The thickness of the sheet is 0.4 um, and the length of the sheet may be modulated from 20 um to 70 um.
• Objectives: Illumination is performed by a custom-made 28X, 0.71 NA long working distance water immersion objective. Imaging is performed by a high-resolution 25X, 1.1 NA long working distance water immersion objective (62.5 X total magnification).
• Microscope: Custom frame microscope made by Intelligent Imaging Innovations (3I). Images are collected using the 3I Slidebook software.
• Cameras: Two 2K x 2K sCMOS Hamamatsu Orca-Fusion camera, 6.5 um pixels, 80% QE, speed up to 100 fps.
• Lasers: Four high-power 500 mW lasers (405 nm, 488 nm, 560 nm, 642 nm).
• Accessories: OKO lab stage insert with temperature and CO2 control. ACQUISITION COMPUTER: Dual 10-Core Xeon 2.4GHz processors, 128GB RAM, NVIDIA Quadro P4000 8GB workstation graphics card, 1TB OS SSD and 6TB SSD array, 10GbE (copper) Adapter; ANALYSIS COMPUTERS: Dual 10-Core Xeon 2.4GHz processors, 256GB RAM, NVIDIA Quadro P6000 24GB workstation graphics card, 1TB OS SSD and 6TB SSD array, 10GbE (copper) Adapter

MINFLUX (MINimising Fluorescence FUXes) nanoscope

• Capabilities: MINFLUX (Abberior) is capable of the two-color long-time imaging with nanoscopic super-resolution (up to 3 nm in XYZ) with low photodamage and photobleaching. This system may be used (a) for molecular mapping of the intracellular structures such as organelles, high molecular complexes, (b) nanometer-scale ultrafast microsecond dynamics of individual molecules associated with intracellular targets such as DNA, transcription factories, nuclear pores, (c) fast micrometer-scale millisecond dynamics of highly mobile (diffusing, gliding, translocating) molecules. This microscope also is capable of confocal and wide-field epifluorescence imaging.
                          MINFLUX technology is based on excitation of the fluorescent molecules with doughnut-shaped beam, and photon-counting. Exact position of the molecule is detected by a minimum of fluorescence; thus, it requires low photon count and imaging is fast and non-photodamaging. Resolution is not dependent on wavelength and molecular tilt.

• Imaging Modules: Olympus IX83-based inverted system from Abberior. Quadscan beam scanner, Ultra-fast beam scanner for MINFLUX, Deformable mirror for precise and fast z-positioning of all beams in MINFLUX imaging, Active sub-nanometer sample stabilization in XYZ, beam auto-alignment package. Acquisition computer with Intel Core, 16GB RAM and GTX670 graphics and Inspector software package.

• Objectives: 100X / 1.45 oil with 0.13 WD for viewing, widefield, confocal and MINFLUX; 60X/ 1.42 oil, 40X/ 1.4 oil, 20X/ 0.8 air, 10X/ 0.4 air.

Detectors: Two filter-based single-photon counting APD detectors for MINFLUX, three spectral APD detectors for confocal imaging with a Rainbow spectral detection tunable from 400 nm to 800 nm, Hamamatsu ORCA-FUSION sCMOS camera 4.0 for epifluorescence.

• MINFLUX Light source: Beam-shaping modules for lasers of 561 nm and 642 nm.

• Confocal Light source: Lasers, 405 nm, pulsed 485, 561, 640 nm.

• Epifluorescence light source: LED Four Color 405/470/590/625 nm.

• Accessories: OKO lab stage insert with temperature and CO2 control.
MINFLUX (MINimising Fluorescence FUXes) nanoscope.