Confocal Microscopy Lab

Overview

Welcome to the Confocal Microscopy Lab at RIT. This lab provides faculty and students with a multidisciplinary imaging research and training facility centered around confocal microscopy.

The laboratory's core mission is to enhance research capabilities, provide an environment that promotes multidisciplinary collaborations, and involve undergraduate and graduate students to support their training and preparation.

The Confocal Microscopy Lab is located in Gosnell A334.

Lab Activities

  • Provide a multidisciplinary confocal microscopy research and training environment to enhance student/faculty interactions and providing a resource to the general community.
  • Provide workshops to train new faculty and student users.
  • Supporting confocal imaging in current courses to expose RIT students to state-of-the-art instrumentation.
  • Providing a multidisciplinary research-based course to foster new research ideas and collaborations and to facilitate the integration of research and education.

Leica SP5

  • Inverted Stand DMI 6000Leica TCS SP5 II Confocal Microscope
  • Motorized Encoded Stage for Mark&Find and Tiling
  • Advanced Optical Module VISIR with Scan Rotation
  • Optical Outfit with EL6000 Illumination & DIC
  • Super Galvo Z Stage for Live XZ Imaging and UltraFast Z Stacks
  • Transmitted Light Detector for Brightfield Confocal
  • Conventional Galvo Scanning System
  • Resonant Scanning System for Live Imaging
  • Five Channel Simultaneous Confocal Imaging
  • Four PMT Detectors One Hybrid Detector with High QE
  • Acousto Optical Beam Splitter (AOBS) Technology
  • AOBS Provides Fully Automated Confocal Imaging with Ultra High Sensitivity
  • RGB Lasers with Total 7 Lines: 458, 476, 488, 496, 514, 542 and 633nm
  • UV 405 Laser with AOTF
  • Premium Workstation
  • Bread Board Top Air Table with Compressor
  • Stage Top Incubation Tokai Hit System

Download Leica TCS SP5 User Manual.

Science Lab, the knowledge portal of Leica Microsystems, offers scientific research and teaching material on the subjects of microscopy. The content is designed to support beginners, experienced practitioners and scientists alike in their everyday work and experiments. Explore interactive tutorials and application notes, discover the basics of microscopy as well as high-end technologies – become part of the Science Lab community and share your expertise! 

DMI6000 Fully Automated Inverted Microscope and Components:

  • Fully Automated DMI 6000 Inverted Microscope "intelligent" Stand
    • 6 Position Fluorescence Axis Motorized
    • 6-fold Objective Nosepiece Revolver M25 thread
    • 8-fold Auto Fluorescence Intensity Module (FIM)
    • Tilting Ergonomic Binocular Tube
    • Scanner Port Left and Additional CCD Port Right
    • Illumination intensity and diaphragms set automatically to selected objective
    • Single keystroke contrast switching BF/POL/DIC/Ph/Fluo
    • 7 Freely programmable buttons to simplify User Specified Tasks
    • Motorized 5-fold coarse and fine focusing with sensitivity and parfocality memory
    • Control Box CTR 6500 with Interface for the transfer of microscope parameters to the SP5
  • Baseplate DMI 6000
  • Universal Mounting Frame
    • Petri Dishes with 24-68mm Diameter
    • Glass Slides with 24-120mm Length
    • Outer Dimension: 160x110 mm
  • Z Galvo Insert for Petri Dishes, 36mm
  • Motorized XY 3-Plate Stage
    • Mark & Find—multipositional timelapse, marking and returning to fields of interest, etc.
    • Tile scanning –automated juxtaposed field acquisition to create a "Montaged" superimage
    • Integral software control within LASAF
    • Mechanical control via STP6000 and/or Smart Move remote control devices field acquisition
  • Optical Outfit DMI 6000 with DIC Components
    • Automated switch btw eyepieces, photo/video or TCS SP5 II port
    • Manual 3-Plate Mechanical XY Stage with holes for mounting Super Galvo Z Stage
    • Pair of adjustable eyepieces HC PLAN s 10x/25
    • Motorized Revolver for Objective DIC prisms
    • Motorized TL Condenser for Automated DIC
    • Condensor S28 with 28mm Working Distance and 0.55 NA
    • Filter system ICT, Analyzer and Polarizer for DIC
    • EL6000 External Fluorescence Light Source, 2000 hour bulb life, Alignment Free, Fiber Coupled to microscope for thermal stability
  • Standard Cubes for Eyepiece Viewing
    • Long Pass DAPI Cube, UV-Excitation (BP 340-380, LP 425)
    • Long Pass GFP Cube, Blue-Excitation (BP 450-490, LP 515)
    • Long Pass RFP Cube, Green-Excitation (BP 515-560, LP 590)

Highest Grade Objectives for Confocal Imaging:

  • Objective HCX PL FL 5X/0.15 Dry
    • FWD 12mm
    • Cover Glass (not critical)
  • Objective HC PL FL 10x/0.30
    • Coverglass 0.17 mm (#1.5)
    • FWD 11.00 mm Objective HC PL FL 20x/0.50
    • Cover Glass 0.17mm (#1.5) -FWD 1.15mm
    • DIC with K3 & D Prisms
  • Objective HCX PL APO 40x/1.1 W
    • Cover Glass 0.17mm (#1.5)
    • FWD 0.63mm
    • High Transmission through 3601100nm
    • DIC with K7 and E Prisms
  • Objective HCX PL APO CS 63X/1.400.60
    • Oil
    • FWD 0.1mm
    • Cover Glass 0.17mm (#1.5)
    • DIC with K10 and E Prisms

DIC Prisms for 20x, 40x and 63x Objectives:

  • IC condenser prism K3
  • ICT condenser prism K 7
  • ICT condenser prism K 10
  • IC Prism D IC Prism E

Super Z Galvo Stage

  • Live XZ Plane Image Acquisitions
  • Z-stack acquisitions at up to 15 frames per second at 512x512 image resolution on RS-Scanner
  • Retains compatibility with standard DIC prisms unlike piezo-focus devices
  • 1500um travel
  • 3nm stepsize
  • 40nm reproducibility

Leica TCS SP5 II AOBS HyD TS System and Components

  • AOBS Based Tunable Filter Free Spectral Confocal
  • GaAsP Hybrid Detectors and PMTs
  • Tandem Scanning with Conventional Galvo and Resonant Scanners
  • Optical Module VISIR with Scan Rotation:
    • Optical coating VISIR with high transmission of > 99% throughout the visible and infrared spectrum ranging from 400-1300nm
    • Low reflection at optical surfaces throughout the visible and infrared spectrum
    • Independent, optical rotation of the scanfield up to around 200° by a highly transparent Abbe-König rotator design
    • Prepared to include either a standard or tandem scanner with Leica parallax-free patented three-mirror design
    • Prepared for use with 405 nm line laser, all line lasers with visible light, white light laser, infrared lasers and optical-parametric oscillator (OPO)
  • AOBS Confocal Module
  • TCS SP5 II Confocal Module with AOBS
    • Ultra High Sensitivity
    • Filterfree Spectral Detection with User Defined Bandwidth
    • Simultaneous use of up to 8 Lasers from UV to IR
    • Replaces the Conventional Rotating Dichroics with Fixed Optical Characteristics
    • Minimum User Supervision with Optimal Flexibility and Automated Design of the Optical Path & Bandwidth
    • Unmatched Flexibility: Mathematically equivalent to 40,320 Dichroics
    • Possibility of "All" combinations of the Excitation and Emission Wavelengths
    • Steep Transmission Cutoff, <2nm
    • Unmatched Spectral Integrity: Flat Spectral Behavior Results Undistorted Recording of Emission Spectra
    • Drastic Reduction in Phototoxicity with Low Intensity and Exquisite Signal Efficiency
    • Facilitates Better Time-Lapse of Living Cells
    • Low Maintenance with "No" Moving Mechanical Parts
    • Five Spectral Detectors with "Independently" adjustable Gain
    • Maximal Fluorescence Detection with Patented Prism Design
    • Automatic Adjustments of Pinhole Diameter
    • Manually Adjustable Pinhole for Improved Z Sectioning
    • Beam parking for FRET and FRAP
  • 2100 Tandem Scanning System SP5 II Conventional Scanner:
    • Up to 2,800 lines-per-second
    • Up to 5 sustained frames-per-second at 512x512 image resolution
    • Up to 25 sustained frames-per-second at 512x16 image resolution
    • Beam-parking -Up to 8192x8192 image resolution
    • 2.12 mm Scanfield diameter with 10x
    • Rotation, panning, zooming capability
  • Resonant Scanner:
    • Up to 16,000 lines-per-second
    • Up to 30 sustained frames-per-second at 512x512 image resolution
    • Up to 300 sustained frames-per-second at 512x16 image resolution
    • Up to 1024x1024 image resolution
    • 1.25 mm Scanfield diameter with 10x
    • Rotation, panning, zooming capability
  • 5-Channel Leica "SP" Spectral Fluorescence Detection
    • Simultaneous detection of up to 5 fluorophores from 400nm to 800nm using prism mirror design for superior transparency (95%) and efficiency
    • Set emission bandwidth as small (specific) as 5nm and as large (efficient) as 300nm e.g. ECFP emission peak at 475nm480nm, broad-spectrum DAPI at 400nm-700nm
    • Set detection window wavelength minimum and maximum to the nanometer eg. FITC alone at 495nm-530nm or simultaneous FITC and TRITC at 495nm-514nm and 563nm611nm respectively.
    • Spectral imaging (ie. separation) of spectrally similar yet intensity dissimilar fluorophores through individual adjustment of amplification gain (voltage) control on MULTIPLE detectors eg. CFP-YFP, GFP-YFP
    • Separation of fluorophores without reference spectra in most cases (simultaneous - optical, sequential-optical, or channel-mathematical)
    • Compatible with fast-RS and TS-scanners for FAST spectral imaging!
  • Transmitted Light Detector for DMI 6000
    • Auto Switching Mirror Confocal & Eyepiece
    • BF/DIC/POL/Phase Compatible (not for MP)
  • Four Confocal Imaging Detectors
    • High Sensitivity Hamamatsu 9624
    • Actively Cooled for Extremely Low Noise
    • Low Dark Current
    • Extended Spectral Range with Increased QE
  • Adaptor Kit for HyD SP Spectral Detectors
  • Hybrid GaAsP Detector for Spectral Imaging
    • UltraSensitive Photon Detection with Maximum Quantum Efficiency
    • QE 2-3 times more than Standard PMTs
    • Broad dynamic range (16 bit)
    • Extremely low noise for increased S/N
    • True Photon Counting Capability
    • Improved Sensitivity Creates Very Low Photo-Toxicity and Improved Quality of Z Stacks
  • Imaging Laser RGB for AOBS
    • R = "red" 10mW HeNe 633nm
    • G = "green" 1mW HeNe 543nm
    • B = "blue" 65mW multiline Argon (458, 476, 488, 496, 514nm)
  • Laser Coupling for UV 405 nm Laser
    • Fiber Coupling, Optical and Mechanical Coupling Interface for UV laser
    • UV Adaptation Optics with Motorized 6-position Revolver
    • Chromatic Correction Device
    • Illumination Pinhole
    • Motorized turret for UV-Pinhole-Optics
    • Simultaneous use of all Lasers is possible
    • Switching between different types of microscopy stands is possible
  • AOTF (AcoustoOpticalTunableFilter) UV/405 nm
  • Laser 405 nm, 50mW Laser System
  • CorrL 405 20x/0.70 CS
    • Unique to SP5 II for UV Correction
  • CorrL 405 40x/1.10 W CORR CS
    • Unique to SP5 II for UV Correction
  • CorrL 405 40x/1.30 OIL CS
    • Unique to SP5 II for UV Correction
  • CorrL 405 63x/1.400.60 oil CS
    • Unique to SP5 II for UV Correction
  • Workstation PLUS with 2x19" LCDs
  • Multitasking HighPower Pentium Workstation with Windows 7 (64Bit) operating system
    • 12 GByte RAM
    • 160 GByte SATA hard disc drive
    • 1000 GByte SATA hard disc drive
    • Intel sixCore Xeon X5650 2.66 GHz
    • 2 TByte SATA Hard Disk Drive
    • 2x eSATA Interface
    • Dual Layer DVDWriter, DVD +/RW -10/100/1000 Ethernet Controller
    • Keyboard and Mouse
    • Two 19"LCD flat screens True colour, 1280x1024 Pixels
    • MSWINDOWS 7
    • Leica LAS system software for control of scan process and image processing
    • Online software and hardware manual
  • Computer Table
  • SP5 Control Panel for Ergonomic Control of Multiple Confocal Functions
    • Panel Box with 6 Freely Programmable Digital Potentiometer Control Knobs
    • LCDs Display Current Assignment and Setting for Each Knob
    • Facilitates easy adjustment of scan parameters without taking eyes off image display
  • LAS Live Data Mode
    • Recording of Manual and Automatic Workflows
    • Allows Correction of Background Fluorescence
    • Complex Time Lapse Measurements
    • Trigger Functions
    • Stack Profiling or ROI measurements
    • Bleaching Experiments and Online Display
  • 3D Reconstruction & Animation
  • LAS MicroLab Software Packages:
    • FRAP Wizard:
    • Step by Step Protocol
    • Efficient Bleaching by Automatic Zoom
    • Quantify the Measurement Data, On- and Offline
    • Export Data in *.xml format FRET AcceptorPhotobleaching
    • Step by Step Protocol for FRET
    • FRET-efficiency will be calculated for user-defined regions and the overall result will be displayed as a FRET efficiency map
    • Export Data in *.xml format.
    • User Guidance with Background Correction and Cross-Talk Removal
    • On-and Offline Quantifying of FRET Efficiency
  • SP5 Microscope Air Table Breadboard Top
    • 30" X 36" Stainless Steel Top, 4" Thick w/ M25 Holes
    • Custom Leg Set, 29" Height (matched to SP5 Table)
    • Casters for easy set up and positioning **Compressed Air Required Air Compressor for Table
  • Tokai Hit GSI Chamber System for SP5 Super-Z Galvo Stage
    • GSI Stage Top Chamber
    • Objective heater
    • Heated Glass Lid
    • 3 Channel Temperature Controller
    • 1 Channel Gas Flow Controller (premix)
    • 35mm Dish Holder
  • GSICGC Dish Attach for Chambered Coverglass

Imaging Software

Biosafety Policies

Please read and review all information provided before you use the facilities.

Application

Once you have read the information and are ready to begin imaging your project, please fill out provided application and e-mail to Hyla Sweet hxssbi@rit.edu. Once the application has been processed an imaging session can be scheduled.

Download Application

Acknowledgement

The RIT confocal microscopy lab is supported through the National Science Foundation Major Research Instrumentation Program (#1126629), the RIT Office of the Vice President for Research, the Kate Gleason College of Engineering, and the RIT College of Science. All images on this site were produced in the CML.