Confocal Microscopes

The RS-G4 has been designed with a compact, high-speed resonance scanner and scanning stage with macro and micro-imaging optics that allow the acquisition of in-vivo and ex-vivo images from slides to cleared whole tissue mounts in the micrometer to centimeter scales.

In addition, ex-vivo tissue sections can be imaged in both reflectance and fluorescence modes. Its 360-degree rotation of the scan head allows greater flexibility and precise angling for acquiring images of large tissue, organs, or whole animals. With a single nose piece, the instrument is essentially agnostic as to what type of objectives can be used. We routinely use our Nikon Plan Apo 10xC/0.5 NA (WD: 5.5 mm) and the Nikon CFI90 20xC/1.0 NA (WD: 8.2 mm) glycerol immersion objectives for imaging cleared tissue.

References

• Ribbon scanning confocal for high-speed high-resolution volume imaging of brain

• High-Resolution, Three-Dimensional Reconstruction of the Outflow Tract Demonstrates Segmental Differences in Cleared Eyes

Equipment Specifications

We have objective options from 1x to 100x, including the Nikon CFI90 20xC/1.0 NA (WD: 8.2 mm) objective designed for cleared samples. Resolution is determined by which objective is used with individual frames (1024 x 1024 pixels) captured at up to 25fps.

Highlights

• Supports air, glycerol, water, and oil immersion objectives
• Fast, large-area mosaic creation up to 120 x 80 mm x 11mm
• Simultaneous and sequential imaging for reflectance and fluorescence
• Experiment acquisition setup for snapshots, mosaics, time, Z-series, and multi-points. wavelength

Lasers

• Wavelengths include: 405 nm, 488 nm, 561 nm, 638 nm, 785 nm

Objectives

Leica DM6000 CFS upright multiphoton microscope equipped with a TCS SP5 MP laser confocal with both galvonomic and resonance scanners and with internal spectral HyD detectors. 405, 458, 476, 488, 514, 543, 594, and 640 nm single photon laser lines and a Mai Tai eHP DS 690-1040 nm tunable multiphoton laser along with 4-channel non-descanned detectors. Objectives available include 63x/NA 1.4 oil and 25x/NA 0.95 water (optimal for intravital deep tissue imaging (up to 2.5 mm)).

Objectives

Features include Nikon's patented PFS Perfect Focus III focus lock technology. LED Illumination systems for both transmitted and reflected light applications. New Lambda anti-reflective coated objectives at 4x, 10x, 20x, 20x water immersion, 40x oil, and 60x oil magnifications for DIC. Fluorescence illumination and filters for widefield applications for DAPI, CFP, GFP, YFP, RFP, and Cy5. The scope is equipped for widefield imaging using a Hamamatsu Flash 4 V3 CMOS camera. Automated collection and analysis with Nikon Elements JOBS is available.

• A1R GaAsP Confocal System
• Laser Selection: 405 nm, 445 nm, 488 nm, 514 nm, 561 nm, and 640 nm.
• The Picoquant Fluorescence Lifetime Imaging Microscopy (FLIM) system has 405 nm, 485 nm, and 560 nm pulsed lasers and can perform many FLIM and FCS techniques.
• A1R Confocal Scanner: Nikon's exclusive hybrid scanner allows flexible switching of two galvo scanners (resonant and non-resonant), this allows ultrafast imaging (up to 420fps) and photoactivation imaging required to unveil cell dynamics and interaction.
• Detectors: Two GaSaP detectors, two enhanced PMT detectors, one transmitted light detector, one Hamamatsu Flash 4.0 V3 CMOS camera
• Configured for automated screening of plates and slides
• A modular system for assay and analysis development (Nikon Elements and JOBS)
• Environmental control for live cell imaging

References

• Fluorescence Lifetime Measurements and Biological Imaging
• Time Correlated Single Photon Counting Wiki

Objectives

* Some objectives have a correction collar for various coverslip thicknesses. See staff for appropriate adjustments.

Features include Nikon’s motorized Ni microscope body. The CFI60 objectives for the Ni system are 10x, 20x, 40x oil, and 60x oil, all with DIC capabilities. An LED illumination system for fluorescence, and filters for widefield using DAPI, GFP, and Tex Red filters. There is a Prior motorized x/y stage and color camera for tiling of large areas of interest in brighfield, DIC, fluorescence, and confocal modes.

• There are 405 nm, 488 nm, 561 nm, and 640 nm laser lines for excitation
• Dual GaSP detectors with continuous filters for spectral imaging

• Three photo-multiplier tubes (PMTs), plus transmitted light detectors, Nikon DS-U3 color camera
• Transmitted Light Filters: ND2, ND4, ND8, ND16, Green, and Cool Blue
• Widefield image capture, including tiling in brightfield, DIC, DAPI, AF488, AF568 and CY5 fluorescence
• Includes Elements modules for 2D and 3D Tracking and Measurements, Ratio and FRET imaging

Objectives

Nikon A1RMP confocal is connected to an upright Nikon FN1 microscope featuring a Plan Apo LWD 25x water immersion, NA 1.1 objective most suitable for imaging deep (it has a 2 mm working distance) into a fixed or living sample. In addition to laser scanning confocal imaging, conventional, and resonant visible laser confocal imaging, a Spectra-Physics 15W Mai Tai eHP tunable IR laser and six, three GaAsPs (epi direction) and three conventional PMTs (trans direction) non-descanned GaAsP detectors allow multiphoton visualization of multiple fluorophores located deep within the specimen. Visible lasers include 405, 457, 488, 514, 561 and 637 nm lines. The system has both galvonomic (4K) and resonant (1k, 8Khz, up to 720fps) scanners that can be used in concert simultaneously for photoactivation and MP imaging. A encoded Prior motorized stage and 200 µm Piezo objective motor can be used for tiling or multiple-position imaging.

The system is also equipped with Second Harmonic Generation (SHG) detection.  SHG doesn't involve the excitation of molecules like other techniques such as fluorescence microscopy. See UIC staff for training and questions.

This instrument was funded in 2012 by the Minnesota Partnership in conjunction with the Mayo Clinic Foundation, Rochester, MN.

In October 2018 the system was upgraded to an A1RHD with 1024 resonant scanning and improved signal-to-noise imaging in resonant mode.

Objectives

Filters - MP

Filters - MP NDD

Nikon Ti-E Motorized Microscope featuring PFS Perfect Focus III focus lock technology. LED Illumination systems for both transmitted and fluorescence light applications. Objectives at 10x, 20x, 40x, 60x (water and oil immersion), and 100x magnifications for DIC. Fluorescence accessories for widefield applications for DAPI, GFP, RFP, and Cy5. The Nikon Elements software has the JOBS package that can automate image collection for high-content screening of plates.

SIM Structured Illumination System-Using high-frequency Structured Illumination, the Nikon N-SIM can achieve an image resolution of 120 nm, which was previously considered impossible with optical microscopes. Furthermore, with a temporal resolution of up to 0.6 sec/frame, N-SIM enables super-resolution time-lapse imaging capture of dynamic molecular interactions in living cells.

• Lasers (A1R): 405 nm, 488 nm, 561 nm, 640 nm
• Lasers (N-SIM): 405 nm, 488, nm, 561 nm, 640 nm
• Detectors: The A1R has four PMTs for confocal detection, two of them GaAsPs, with double the sensitivity of traditional PMTs. There is also a transmitted light detector and a 32-channel spectral detector.
• N-SIM: Hamamatsu FLASH 4

Objectives

Olympus FluoView FV1000 is designed for high-resolution, confocal observation of both fixed and living cells. The FV1000 offers advances in confocal system performance while providing the speed and sensitivity required for live cell imaging, with minimal risk of damage to living specimens. The system has four fluorescence detectors plus a transmitted light detector for DIC. 

• Automated upright  BX61 microscope base
• 100W Mercury fluorescent lamp
• 100W halogen transmitted illuminator with detector
• PRIOR ProScanII motorized XY stage 
• DIC optics

​Methods

• Fluorescence, Brightfield, DIC, FRAP, FRET

​Automated Capabilities

• Time-lapse, Multi-channel fluorescence, Co-localization, Z-stacks 

Lasers

• 405nm 25mW Diode, 458, 488, 515nm 30mW Argon, 543nm 1mW HeNe green, 635nm 10mW HeNe red

Detector 

• 4 photomultiplier tubes channels, 1 transmission detector

Objectives

Filters

Channels

This system is equipped with pulsed 405 and 485 nm lasers for FLIM. FLIM is the acronym for "Fluorescence Lifetime Imaging Microscopy" and is the technique that maps the spatial distribution of fluorescence lifetimes within microscopic images of fixed or living cells. The fluorescence lifetime measures the time that a population of fluorescent molecules spends in the excited state. The fluorescence lifetime does not change upon intensity variations and therefore lifetime measurements are not dependent on the local concentration of fluorophores, or variations in the optical path of the microscope, the local excitation light intensity, or on the local fluorescence detection efficiency.

The FLIM is a time domain system and uses pulsed lasers at 405 and 488 nm. The detector on the system is a Hybrid GaAsP cathode with three possible emission filter setups - a 480/30nm bandpass for CFP emission, a 460LP, and a 525/50 nm bandpass filter for GFP. 

Automated Capabilities

• Time-lapse, Stitching, Multi-Channel fluorescence, Co-localization, Spectral unmixing, Z-stacks

External FLIM

• PicoQuant pulsed lasers at 405 and 485 nm
• Software includes Becker&Hickl SPCM and SPC Image

Lasers 

• 405nm 25mW Diode
• 458, 488, 515nm 30mW Argon
• 543nm 1mW HeNe green
• 635nm 10mW HeNe red

Detector

• 3 photomultiplier tubes channels
• 1 transmission detector

Objectives

Filters

Channels

Zeiss Cell Observer Spinning Disk Confocal

The Zeiss Cell Observer Spinning Disk confocal fluorescence microscope for imaging live samples at fast speed. Definite focus, time-lapse, multi-well, and tiling of images. DIC and phase contrast capable. Full environmental chamber for temperature and atmosphere control. Three laser lines are available (405, 488, and 561 nm). 

Motorized Zeiss Axio Observer Z1 inverted scope with temperature-controlled enclosure, CO2, Definite Focus, multi-channel acquisition, piezo x/y/z stage.

Methods

• Fluorescence, Brightfield, Phase contrast, DIC, Time Lapse, Live Cell with 37°C / CO2 enclosure

Automated Capabilities

• Time-lapse, Stitching, Multi-channel fluorescence, Co-localization, Piezo Z-stacks

Lasers

• 405 nm, 50 mW, 488 nm, 100 mW, 561 nm, 30 mW

Detector

• Photometrics QuantEM 512SC - High Sensitivity 16 bit, 512 x 512 grayscale CCD

Objectives

• Nikon NiE fully motorized (multi-position nosepiece, filters, condenser, z-motor, x/y stage) upright microscope with 25 mm field of view. This resonant (15 fps full FOV with up to 720 fps at 2048x16 pixels) and galvanomic scanners (up to 8192x8192 pixels for Nyquist resolution at lower magnifications or larger FOVs)
• Lasers and detection filters utilizing the  408, 445, 488, 514, 561, and 640  nm excitation lines allow for multiplexed imaging of DAPI, CFP, YFP, GFP, FITC, dsRed, TRITC, CY5, and many more fluorescent reporters.
• Four-channel GaAsP PMT detector modules, including spectral detection along with transmitted light.

• Objectives 2x, 4x, 10x, 20x, 40x, 60x, 100x including water, silicon and oil immersion options. The system is compatible with many of the UIC’s extensive collection of additional Nikon objectives.
• Differential Interference Contrast (DIC)
• Nikon Element ER software has built-in multidimensional (multi-XY, Z, T, multichannel) experiment capabilities. The JOBS module allows customization in setting up non-orthogonal experiments with multiple paths and dimensions. Analysis of data can be done in real-time during the experiment, and the direction of the experiment can even be changed based on the results of the analysis. Fully automated collection and analysis capabilities with JOBS. The UIC staff is excited to help train you from the most basic image acquisition to the full complement of our instrument's capabilities.

The Nikon A1si is a confocal system equipped with a point-scan confocal scanner, four PMT detectors for fluorescence and one for transmitted light. The scope also has a 32-channel PMT spectral detector. The A1si's highest pixel resolution mode is up to 4096 x 4096 pixels. The system is mounted on a Nikon Eclipse Ti2 inverted scope equipped with Perfect Focus System, fluorescence filters with DIC optics and a widefield Hamamatsu Flash 4 camera. There is also an IBIDI environmental stage top incubator system for long-term live cell imaging. Nikon NIS Elements imaging software is used to control acquisition and analysis. Nikon JOBS will permit investigators to create complex automated image acquisitions with intelligent analysis.

The advantages of multispectral imaging and unmixing have been shown in hundreds of publications to reduce autofluorescence by as much as 100-fold, both for microscopy and FFPE tissues and in small animal and plant fluorescence imaging.

Lasers 

• 405 nm diode at 38mW, Argon multiline laser 457/477/488/514nm at 40mW, 561 nm diode at 40mW, 638 nm diode at 10mW

Detectors

• 4 PMTs, a Transmitted light detector, and a 32-channel spectral detector

References

Tam, et al "Improved in vivo whole-animal detection limits of green fluorescent protein-expressing tumor lines by spectral fluorescence imaging" Molecular Imaging, 6, 2007, 269.

Mansfield, et al "Visualization of microscopy-based spectral imaging data from multi-label tissue sections" Current Protocols in Molecular Biology, 2008 Oct; Chapter 14:Unit 14.19. DOI: 10.1002/0471142727.mb1419s84.

Levenson, et al "Multispectral imaging in biology and medicine: slices of life." Cytometry A. 2006 Aug 1;69(8):748-58.

Objectives

Filters

Widefield Filters

Confocal Filters

The AZ100 Multizoom Macroscope with the C1si Spectral Confocal attachment makes the ultimate platform for in vivo imaging. Ideal for plant biology, developmental biology, cell biology, stem cell, and tissue research, the AZ-C1si allows the researcher to view large specimens in confocal mode. The AZ-C1si can capture fields of view up to 1cm and permits deeper confocal imaging than conventional microscopes thanks to its large working distance objectives. Whole organisms can be monitored and documented over time (for example, embryos) offering a wealth of continuous information on development or the organism’s response to experimental variables. Both conventional PMTs and Spectral detectors are installed on this instrument.

The system is equipped with 1x, 4x, and 5x objectives with variable optical zoom.  There are 405, 457, 476, 488, 514, 561, and 637 nm lasers for image, co-localization, FRAP, FRET, and spectral studies.

The system is run using Nikon Elements Software.

The advantages of multispectral imaging and unmixing have been shown in hundreds of publications to reduce autofluorescence by as much as 100-fold, both for microscopy and FFPE tissues and in small animal and plant fluorescence imaging. 

Lasers

405 nm, 457 nm, 488 nm, 514 nm, 567 nm, 637 nm

References

Tam, et al "Improved in vivo whole-animal detection limits of green fluorescent protein-expressing tumor lines by spectral fluorescence imaging" Molecular Imaging, 6, 2007, 269.

Mansfield, et al "Visualization of microscopy-based spectral imaging data from multi-label tissue sections" Current Protocols in Molecular Biology, 2008 Oct; Chapter 14:Unit 14.19. DOI: 10.1002/0471142727.mb1419s84.

Levenson, et al "Multispectral imaging in biology and medicine: slices of life." Cytometry A. 2006 Aug 1;69(8):748-58.

Objectives

Filters

Channels

Leica DMi8 inverted laser scanning confocal microscope with both galvonomic and resonance scanners and 5 spectral internal HyD-S detectors.  A fixed 405nm violet laser and pulsed white-light laser (WLL) that is tunable from 440-790nm allows for precise excitation control and multi-spectral imaging.  The WLL and HyD-S detectors allow for Leica's patented Tau gating and separation modalities using fluorescence lifetime measurements.  Environmental control enables long-term live cell imaging and the Leica Navigator interface streamlines stage control and multi-position high-speed tiling. 

Objectives include 20x/0.7NA multi-immersion, 40x 1.25NA glycerol motCORR, 63x 1.4NA oil, and 63x 1.2NA water.