Imaging

  • Fee for imaging services for internal and RI-EPSCoR users for FY2015: $7.50 / hr.
  • Please see our Fees page for instructions regarding payment for services.
  • All users must complete a Sample Identification Form before initial use of the Zeiss imaging systems.  Users must also read and comply with the Rules for Safe Conduct before using the imaging systems.
  • All users of the Zeiss LSM 700 confocal system must receive Laser Safety Awareness training administered by the RIGSC manager before using this component.  Please refer to the RIGSC Laser Safety Policy for more information.
  • Users who intend to examine live Biosafety Level 2 material must follow the Standard Operating Procedure for BSL-2 samples.

ZEISS Axio Imager M2 light microscope

The RIGSC’s imaging systems utilize a ZEISS Axio Imager M2 upright microscope that is equipped for transmitted light microscopy with brightfield, phase-contrast, and differential-interference contrast (DIC) methods.  The Axio Imager M2 is also set up for epifluorescence microscopy using an HXP 120 metal halide lamp and filter sets for DAPI, FITC, Rhodamine and Propidium Iodide excitation / emission.

Almost all functions of the Axio Imager M2 are motorized and can be operated through a touch-sensitive TFT display.  The Axio Imager M2 is also equipped with a motorized stage to allow for automated acquisition at multiple locations and tiled montage images with correlated image stitching.

Zeiss Efficient Navigation microscope software (ZEN) is the single user interface for all imaging systems from Carl Zeiss.  ZEN uses a new data format (.czi) that allows processing of large amounts of data acquired with fast 3D imaging systems.  Alternatively, images may be stored as jpeg files or in several other formats including OMETIFF, the image specification of the Open Microscopy Environment including metadata, to facilitate cross-platform image data exchange.  ZEN Blue (formerly AxioVision) is used to control imaging functions utilizing the AxioCam HRc digital camera while ZEN Black is used for control of the laser scanning confocal system, LSM 700.  Two guides are available for ZEN BlueFirst_Steps.pdf and QuickStartGuide_Multidimensional_Imaging.pdf.  A free version of the software to open and process .czi files, ZEN lite 2012, is also available.

Axio Imager M2 Objective Specifications

Objective

Magnification / Numerical Aperture

Working Distance (mm)

Phase Contrast

Differential-Interference Contrast

Plan Neofluar

5x / 0.15

13.6

no

no

Plan Neofluar

10x / 0.3

5.6

no

no

Plan Neofluar

20x / 0.5

2.0

Ph 2

DIC II

Plan Apochromat

20x / 0.8

0.55

no

DIC II

Plan Neofluar

40x / 0.75

0.40

Ph 2

DIC II

EC-Plan Neofluar

40x / 1.3 oil

0.20

no

DIC II

Achroplan

40x / 0.8 water

3.60

no

DIC II

Plan Apochromat

63x / 1.4 oil

0.19

no

DIC III

Plan Neofluar

100x / 1.3 oil

0.20

Ph 3

DIC III

ZEISS AxioCam HRc digital camera

The AxioCam HRc is a high resolution color digital camera that can be used at different resolutions depending on the application: from 1388 x 1040 up to 4164 x 3120, corresponding to 13 megapixels per color channel.  With two different read-out speeds (12.5 MHz and 25 MHz) the HRc is versatile in terms of the range of applications it can perform. With the fast read-out mode of 25 MHz, live images of between 12 images/sec (full resolution) and 33 images/sec (reduced resolution) can be achieved.  The extremely high sensitivity of the large 1.7 cm sensor, an outstanding signal to noise ratio and Peltier cooling for long exposure times allow the HRc to make high-quality imaging possible.  By scanning all red, green and blue components of the image, the patented HRc Color co-site Sampling technique achieves a color brilliance that can otherwise only be accomplished using 3 sensors.

ZEISS LSM 700 scanning confocal imaging system

Unlike classical microscopes, laser scanning microscopes feature a confocal pinhole aperture positioned in the beam path and conjugate with the illumination focus.  Its diameter can be varied to ensure that the detector receives light exclusively from the focal plane.  Light from above or below the focal plane is rejected, which increases the definition of the image.  The confocal microscope scans the specimen in a point-by-point mode thus creating a 2D image known as an optical section.  Several optical sections, taken along the microscope’s Z axis, form a Z-stack, which the software converts into a 3D image with an extended depth of focus.

The high sensitivity of the LSM 700 is guaranteed by the innovative optical design by Carl Zeiss, which conducts the light emitted by the specimen onto the detectors with almost no photon loss.  The light path in the LSM 700 scanning module, from the source to the detectors, is shown below.  Excitation light from up to four lasers is coupled into the scanning module via optical fibers (1). It falls on the beam-combining mirror cascade (2), where it is centered and aligned with the optical axis.  Two scanning galvanometer mirrors (3) direct the light onto the specimen, which is scanned by the light beam in a point-by-point mode.  The fluorescent light emitted by the specimen is contaminated by a small amount of reflected laser light; this is efficiently blocked by the FixGate main beam splitter (4).  The remaining emission light is directed through the fully automatic, high-precision pinhole (5), which exclusively allows fluorescent light originating from the objective’s focal plane to pass. This reaches another beam splitter (6), where it is split up and directed onto the two detectors PMT1 (7) and PMT2 (8).  Filters (9) may optionally be positioned in the beam path between the VSD beam splitter and the detectors.

The LSM 700 optics are designed for high transmission and diffraction-limited imaging over the spectral range 350–800 nm.  All lasers are fiber coupled to the scan head and the optical fibers are directly coupled to the laser output windows for long-term stability.

LSM 700 Laser Specifications

            • Violet, 405 nm (DAPI), 5 mW, LED laser class 3B
            • Blue, 488 nm (FITC), 10 mW, LED laser class 3B
            • Yellow, 555 nm (Cy3), 10 mW, LED laser class 3B
            • Red, 639 nm (Cy5), 5 mW, LED laser class 3B

A transmitted light detector (T-PMT) can be used to acquire non-confocal transmitted light images in bright field, phase contrast or DIC using scanned laser illumination. These images are exactly aligned with confocal fluorescence images and can be acquired simultaneously.

The LSM 700 operating software, ZEN Black, has a wide range of capabilities that include the following features:

  • Configuration of all motorized functions of microscope, scanning module and lasers  Configurable and savable workspace (user interface)
  • Saving and restitution of application-specific configurations (ReUse)
  • System self-test: Calibrating and testing tool for automatic system checking and adjustment
  • Smart Setup: Automatic setting of the system according to a selection of dyes
  • Acquisition modes: Spot, Line/Spline, Frame, Z-stack, Lambda Stack, Time Series and all combinations (XYZ | t)
  • Online computation and presentation of ratio images; averaging and summation (linewise, framewise, configurable), Step Scan (for higher frame rates)
  • Crop function: Convenient selection of scan areas (zoom, offset, rotation simultaneously); RealROI Scan, Spline Scan, scan of up to 99 ROIs of any shape, pixel-precise laser blanking; scan along a freely defined line
  • ROI Bleach: Localized bleaching in up to 99 bleaching-ROIs for applications such as FRAP or Uncaging; use of different speeds for bleaching and image acquisition, use of different laser lines for different ROIs.
  • Multitracking: Fast change of excitation lines when acquiring multiple fluorescences, for minimizing signal crosstalk
  • Lambda Scan: Sequential acquisition of image stacks with spectral information for every pixel
  • Linear Unmixing: Generation of crosstalk-free multifluorescence images with simultaneous excitation; online or offline unmixing, automatically or interactively; advanced unmixing logic with reliability statement
  • Presentation: XY, Orthogonal (XY, XZ, YZ), Cut (3D section), 2.5D for time series of line scans

A quick guide describing the basic operation of the LSM 700 using ZEN Black is also available.

To make an appointment for the ZEISS imaging system, please use the RIGSC Calendar.  If you have any questions or problems, call the RIGSC manager at 874-5919.