Links
The University of Colorado Boulder Library Search Engine is great for locating primary literature and text books for research.
These short courses by iBiology Microscopy are designed for beginners.
Microforum has a wealth of information and is recommended for more advanced users. (Our very own Director is a moderator there!)
There is also some fun microscopy information in the links below:
- Imaris 9 overview, with focus on features of version 9.6
- Imaris - An introduction to 3D visualization & analysis techniques for microscopy data
- Olympus Resource Center
- Nikon's MicroscopyU
- Leica Science Lab
- Zeiss Education
- Multiphoton Excitation Spectra Database (University of Arizona)
- Semrock Optical Filters
- Chroma Technology Corp
- Hamamatsu Knowledge and Hamamatsu Camera Simulator
- iScopeCalc :: Mobile Microscope Calculator
- University of Colorado at Boulder | Geometric Optics Phet Simulation
If you have not stumbled across the University Software Downloads and Licensing site, I highly recommend checking out the software that is available for students and staff.
Either way - here are a few well-known image viewers and advanced computer languages for image processing and analysis:
- Fiji (Is Just ImageJ)
- Vaa 3D
- Imaris Viewer Free 3D/4D Microscopy Imager Viewer
- Olympus cellSens Viewer
- Leica LAS X
- 3i Slidebook Viewer
- Nikon Elements Viewer
- Wolfram Alpha Mathematica (University Site License)
- Matlab (University Site License)
https://www.colorado.edu/lmcf/choose-type-microscopy
Multi-photon microscopy:
‘Gemini’ – Olympus FVMPE-RS multi-photon microscope.
This is a dedicated multi-photon microscope built on an Olympus BX63LF upright frame. The microscope is equipped with two separate, pre-chirped, tunable, pulsed IR lasers, a Spectra Physics Insight X3 (680-1300 nm) and Spectra Physics MaiTai eHP DeepSee (690-1040 nm). The microscope has four PMT detectors, two enhanced and two high-sensitivity GaAsP. All optics are designed for long wavelength transmittance and reflectance. The hybrid scanning system with galvanometer and resonance scanners is ideal for high-temporal resolution imaging. Available multi-photon optimized objectives include a 5x NA 0.10 air WD 20 mm, 10x NA 0.60 water immersion WD 3.3 mm, 25x water immersion NA 1.05 WD 2.0 mm and 60x water immersion NA 1.10 WD 1.5 mm. A 20x air, NA 0.45 WD 8.2 mm is available for GRIN lens work.
This system is controlled with advanced Olympus Fluoview acquisition software, a motorized nosepiece and X-Y Prior Z-Deck stage allowing for multipoint, tiling, Z-series time-lapse imaging, etc. Other features include auto-alignment of the lasers, beam- shaping for improved deep focus, and a motorized correction collar for the 25X objective to correct for refractive index over depth to provide maximum brightness, contrast and resolution through the entire imaged volume. Laser output power can also be software adjusted and tuned through the imaging depth.
A sample warming pad (Stoelting) and isoflurane anesthesia is available for imaging small animals. This microscope is housed in an independent, secured room with significant light mitigation, inside a custom-made dark box. CellSens Dimension with deconvolution (Olympus) and Imaris for Cell Biologists 9.2.1 (Bitplane) software are available on a separate HP Z8 workstation for data analysis. This microscope is excellent for simultaneous multi-color, deep, in situ imaging of excised or live samples, including tissues and whole animals under anesthesia.
Confocal and super-resolution microscopy:
‘Thor’ - Nikon A1/N-SIM with TIRF.
Built on an inverted Nikon Ti-E microscope, the A1/N-SIM is a multi-purpose, inverted laser scanning confocal microscope, TIRF and super-resolution imaging platform. This microscope uses a high-output LUNV6 laser launch. There are six laser lines and up to 4 fluorophores can be imaged nearly simultaneously. Laser lines include: 405, 445, 488, 515, 561 and 647nm.
The A1 confocal uses four PMT detectors, two conventional and 2 high-sensitivity GaAsP, while TIRF or SIM use an ANDOR-897 EMCCD detector. Widefield viewing is illuminated to the eye using a SOLA Light Engine (Lumencor). The microscope uses a motorized X-Y stage, motorized Z-drive, Nikon Perfect Focus system generation 3, and a MCL Piezo Z-stage. Two-dimensional and three-dimensional structured illumination microscopy is available at 60x water immersion and 100X oil immersion. Multi-channel TIRF is available with motorized TIRF angle switching; this is not simultaneous multi- channel TIRF. TIRF is available at 100X. Available objective lenses include: 4x NA 0.2, 10x NA 0.45, 20x NA 0.75, 40x ELWD NA 0.60, 40x NA 0.95, 60x water NA 1.27, 60x oil NA 1.40, and 100x oil NA 1.49 TIRF; DIC is also possible with laser scanning microscopy.
A TOKAI-HIT Cell Incubator with 1x35 mm dish and multi-well slide options is available. Two- and three-dimensional super-resolution structured illumination microscopy (SR-SIM) achieves resolutions typically near 100 nm in X-Y and 300 nm in Z. The microscope is software controlled with NIS Elements v5.21.03. This microscope is excellent for fixed samples, precise optical sectioning, montaging small areas, super resolution imaging, and TIRF.
‘Iron Man’ - Zeiss CLSM510 Confocal Laser Scanning Microscope.
The Zeiss 510 is an inverted laser scanning confocal microscope. It has 7 laser wavelengths and is capable of imaging up to 4 fluorophores simultaneously. The wavelengths include: 405, 458, 477, 488, 514, 543 and 633 nm. This microscope uses standard PMT detectors; widefield viewing by eye uses a mercury arc lamp; Motorized in Z; 5x NA 0.15, 10x NA 0.30, 20x NA 0.80, 40x oil NA 1.30, 63x oil NA 1.40, and 100x oil NA 1.40 objectives are available.
‘Kronos’ – Yokogawa Inverted Spinning Disk Confocal Microscope (Olympus).
The Yokogawa CV1000 is a high-performance spinning disk confocal microscope with an enclosed, light-proof, environmentally controlled box. This microscope has 3 laser lines and is capable of outstanding brightfield microscopy via LED transmission. Laser lines include: 405, 488, and 561 nm. This microscope is equipped with a Hamamatsu high-resolution mode 1024x1024 EMCCD detector and dual Nipkow disks with 50 or 25- micrometer lens pinhole arrays that can be removed from the light path for widefield imaging. The motorized X, Y, Z stage coupled with Olympus’s Zero Drift auto focus system and software focus are great for live cell and high-temporal resolution imaging. Available objective lenses include: 4x NA 0.13, 10x NA 0.40, 20x NA 0.75, 20x LWD NA 0.45, 40x LWD NA 0.60, 40x oil NA 1.30, 60x water NA 1.20, 60x oil NA 1.35, and 100x oil NA 1.40. Three environmentally controlled and atmospheric regulated incubated chambers are available. Chambers include: 1) 1x35mm dish, 2) 3x35mm dishes and 3) low-profile multi-well plates. The microscope is controlled by Yokogawa CV1000 software and is excellent for fixed samples, dim samples, fast dynamic live samples, live tissues, organoids, embryos, multipoint montaging, and low-to-medium throughput screening in 96 or 384 well plate formats.
‘Black Widow’ - Nikon Inverted Spinning Disk Confocal Microscope.
This spinning disk confocal microscope is built on an inverted Nikon Ti-E microscope and is configured with 4 laser lines, which can be used to image up to 4 fluorophores nearly simultaneously. The laser lines are: 405, 488, 561, and 640nm. The microscope has a stand-alone 25 mW OBIS 405 nm photobleaching/photoactivation laser. This microscope uses an ANDOR-897 512x512 EMCCD detector for confocal imaging, and a Hamamatsu 512x512 EMCCD detector is available for widefield imaging. The confocal spinning disk is a Nipkow disk with an array of 50-micrometer microlenses from Yokogawa (CSU-10). A PI Nano Piezo Z-stage is available for rapid three-dimensional imaging. Viewing by eye and widefield uses a SOLA Light Engine (Lumencor).
The microscope is also equipped with a motorized X, Y, Z stage, MCL Piezo Z-stage insert, Nikon Perfect Focus system generation 3 and the following objective lenses are available: 4X NA 0.15, 10x NA 0.45, 20x NA 0.75, 40x NA 0.95, 60x oil NA 1.40, and 100x oil NA 1.45. A TOKAI-HIT Cell Incubator with 1x35 mm dish and multi-well slide option are available. The microscope is software controlled with NIS Elements v4.3 software and is excellent for fixed samples, low light imaging, fast dynamic imaging of live samples, single point photo-bleaching or -activating (405 nm laser), triggered acquisition and multipoint montaging.
Widefield, brightfield and stereo microscopy:
‘Dante’ - Olympus IX81 Inverted Widefield Microscope.
This Olympus IX81 is an inverted widefield epifluorescence microscope. This microscope uses a high-intensity Prior Scientific Lumen 200Pro 200-watt metal halide lamp. Fluorescent imaging is selected using the motorized filter wheel and motorized Prior Scientific excitation and emission filter wheels, with Chroma and Semrock optical filters. The optical filters are standard for imaging QD360, DAPI/Hoechst, ECFP, EGFP/Alexa488, YFP/Venus, Alexa555/568/594;TxRed;mCherry/TdTomato, Cy5/Alexa647, CFP:YFP FRET, other related fluorescent molecules and some custom configurations. The scope is also brightfield and phase-contrast (20x and 40x) capable. The detector is a Hamamatsu ORCA R2 CCD and is equipped with a Prior Scientific H117 motorized, linear-encoded stage (X-Y), Olympus motorized Z-drive, software focusing, multipoint/tiling and the following objective lenses are available: 4x NA 0.13, 10x NA 0.40, 20x NA 0.70, 40x NA 0.75, 60x water NA 1.20, 60x oil NA 1.35, and 100x oil NA 1.40. For live cell imaging, an InVivo Scientific stage-top incubation chamber is available in single 35 mm dishes or multiwall plate formats, using pre-blended atmospheric gas. The microscope is controlled with Slidebook 6.0 software and is excellent for fixed samples and long-term timelapse imaging of live cells.
‘Pepper’ - Nikon Inverted Widefield Microscope.
This Nikon Ti-U is an inverted widefield epifluorescence microscope with both fluorescence and color imaging (histology). For fluorescence, this microscope uses an Excelitas Excite 100-watt metal halide lamp, Chroma optical filters, and is capable of imaging DAPI/Hoechst, EGFP/Alexa 488, TxRed/mCherry/Alexa 568/594, Cy5/Alexa 647 and related fluorescent molecules. Brightfield imaging is available, including DIC microscopy at 60X. This microscope uses a CoolSNAP HQ2 CCD detector for fluorescence. Brightfield-color microscopy is available via a Nikon DS-Fi3 24 bit, high- definition color camera (5.9 megapixel, CMOS). Other features include an ASI motorized X, Y, Z stage and the following objective lenses are available: 4X NA 0.15, 10x NA 0.45, 20X NA 0.50, 40X NA 0.75, 40x NA 0.95, 60x oil NA1.40, and 100x oil NA 1.30. This microscope is excellent for imaging fixed samples, yeast, and other cells.
‘Captain’ America - Leica Upright Widefield Microscope.
This Leica DMRXA is an upright widefield epifluorescence microscope. This microscope uses a Sutter 75-watt xenon lamp, optical filters and is capable of imaging DAPI/Hoechst, EGFP/Alexa488, TxRed/mCherry/Alexa568/594, Cy5/Alexa647 and related fluorescent molecules. This microscope uses a QImaging Retiga 2000R CCD detector, Prior Scientific motorized X, Y and Z stage and has the following objective lenses available: 10x NA 0.30, 20x NA 0.70, 40x NA 0.85, 63x oil NA 1.40, and 100x oil NA 1.40. The microscope is software controlled with Slidebook 6.0 and excellent for fixed samples.
‘Frank’ – Zeiss Fluorescent Stereo Microscope.
This Zeiss Discovery.V12 is a stereo microscope with fluorescent imaging. For fluorescence, this microscope uses an Excelitas Excite Series 120 metal halide lamp, and is capable of imaging EGFP, and TxRed/mCherry. There is also a FOSTEC gooseneck, halogen white light source for specific illumination. A Nikon Digital Site monochrome camera is available for imaging. Total system magnification is variable, from approximately 1.4-80X.
Analysis workstations:
‘Imaris cellSens’ - Hewlett Packard Z8 G4. This analysis PC features Intel Xenon 28 core processors, a 512GB SSD, 4TB HDD N 1 P4317Q data drive, 128GB DDR4 RAM, high-speed USB3.0 ports, and a Dell 43 inch Ultra HD 4K monitor. Software includes, Imaris 9.2.1 for Cell Biologists, Olympus cellSens with 3D deconvolution, FIJI, and CV1000 viewer (Yokogawa).
‘HULK’ – Dell Precision 7910. This analysis PC features Intel Xenon E5-2620 6 core processors, a 512GB Samsung 850 EVO SSD, 2TB HDD, and 8TB SeaGate IronWolf data drives, 32GB RAM, high- speed USB3.0 ports, and dual Dell 24 inch UltraSharp monitors. Software includes, NIS-Elements with SIM reconstruction, MATLAB, Zeiss Zen Lite, Cell Profiler, FIJI, ICY, and CV1000 viewer (Yokogawa).
Image Processing and Data Analysis
- Matlab on Image Processing
- Wolfram Alpha on Images
- Fiji (Is Just ImageJ) - Search Engine
- Life Technologies Molecular Probes School of Fluorescence
- Guides for ethical image processing and various handbooks
Mounting Medium
Facility recommendations:
- Life Technologies SpectraViewer
- Fluorescent Protein Database - FPbase
- CiteAB: Antibody database search engine
- Chroma Spectra Viewer
- Semrock SearchLight
- U Arizona - Spectra Database
- Olympus FluoView Resource Center Dual Probes
- BD Biosciences Fluorescence Spectrum Viewer
- Clontech Fluorescent Proteins
- UNC Michael Hooker Microscopy Facility Table of Fluorochromes
Please learn and follow the Standard Operating Procedures listed below while working in the LMCF.