Dual wavelength imaging without compromise

Conventional methods of dual wavelength imaging such as using split frame optical devices, a rotating filter wheel, or a 3-CCD color camera result in compromises, such as a reduced field of view and others.

But with the ORCA-D2 dual CCD camera, you don’t have to compromise. You can capture dual wavelength images simultaneously, at wavelength ranges of your choice, and with a full field of view in each image.

In addition, the ORCA-D2 camera automatically corrects focus and alignment between the two images to produce images with high precision.

ORCA-D2 is supported by MetaMorph (v.7.3.3), LabView, and Micro-Manager.

Comparison of ORCA-D2 with conventional methods

Dual wavelength imaging method	Pros	Cons
Split frame optical devices	Simultaneous capture at dual wavelengths Selectable wavelengths	Reduced field of view Steps required to correct color or image
Rotating filter wheel	Selectable wavelengths Wide field of view	Dual wavelength images are not captured simultaneously No correction of color or image shifting
3-CCD color camera	Simultaneous capture at dual wavelengths Wide field of view	Exact wavelengths cannot be selected No correction of color or image shifting
ORCA-D2 dual CCD camera	Simultaneous capture at dual wavelengths Selectable wavelengths Wide field of view Auto-correction of color or image shifting

Quick guide to ORCA-D2 camera

To help you determine if the ORCA-D2 is suitable for your imaging needs, wavelength sensitivity and other important parameters are listed in the table below.

Parameter	ORCA-D2 camera
Image sensor type	2 cooled interline CCDs (1.2 megapixels)
Wavelength sensitivity	Visible to NIR
Intensity of signal	Low light
Frame rate at full resolution	11 frames per second
Exposure time	117 microseconds to 60 seconds
Spatial resolution (pixel size)	6.45 microns

The main features and benefits of the ORCA-D2 camera are summarized below. For more info, download the datasheet or contact us by completing the form.

Features	Benefits
Two ER-150 interline CCD image sensors with peak QE over 70% and 6.45 micron pixels	High resolution and short exposure times
Single camera body with two CCDs	Saves bench space and allows easy setup
Interchangeable optical blocks with built-in memory	Rapid filter changes with automatic image alignment
Independent exposure control of each CCD	Very wide dynamic range image pairs with good signal-to-noise ratio in each
Automatic synchronization of dual image readout	Dual image capture is as easy as single image capture
Adjustable, motorized Z-focus of one CCD	Reliable multifocal point imaging
Dual images produced at 11.27 fps	Effective frame rate is over 22 fps with perfect timing at full resolution

Special features

Dual CCD camera structure

The advantages of the ORCA-D2 over conventional dual wavelength imaging methods stem from its unique camera structure. The camera features two CCD image sensors housed in a single camera, and an optical block that incorporates beam splitters and emission filters to separate wavelengths.

Internal structure of camera head. Optical block shown.

Five types of interchangeable optical blocks are available, each with a different wavelength range. See table below for the wavelength ranges.

Dual light modes

The ORCA-D2 has a “high light mode” for handling most applications, and a “low light mode” for when increased quantum efficiency (QE) in the spectrum between visible and near-infrared is required. Within these regions, the CCD’s QE peaks at over 70%, which is the highest level for interline CCDs.

Spectral Response

This sample is typical of the CCD characteristcs, not guaranteed.

Auto-correction features

During dual wavelength image acquisition, the camera will automatically correct image alignment, color mismatch, and other aberrations. Hardware and special software work together to adjust focus and alignment to your experimental setup. Calibration results are saved in the software, eliminating the need to readjust the parameters for the same experimental setup.

Applications

The ORCA-D2 dual CCD camera is suited for dual wavelength imaging in the visible and NIR wavelengths that requires high resolution and an exposure time between 117 µs and 60 seconds. Example applications are listed below.

Ratio imaging
Single and dual wavelength fluorescence microscopy
Fluorescence resonance energy transfer (FRET)
Blue to NIR fluorescence applications
Colocalization and fluorescence in situ hybridization (FISH) applications
Dual wavelength TIRF microscopy
Dual wavelength real-time confocal microscopy
Combined transmission and fluorescence imaging
Multi-focal point imaging microscopy

Sample Images

Ca²⁺ and PKC dual imaging using Fluo-4 and DsRed (optical block: DM 550 nm)

The images to the left show fluorescence signal from Fluo-4 AM loaded cells expressing PKC-DsRed when excited by 480 nm light. Observable in this time sequence is the translocation of PKC-DsRed to the cell membrane concurrent with free Ca²⁺ elevation in response to a depolarizing stimulus.

Sample: Ins-1 cell (insulin-producing cell)
ORCA-D2 optical block: A11400-04 (DM 550 nm, Em1 520 nm/35 nm, Em2 593 nm/40 nm)
Microscope: Olympus IX71
Objective lens: Olympus UPlanAPO 60x / 1.20 W

Samples images courtesy of Hideo Mogami, Ph.D. Dept. of Physiology, Hamamatsu University School of Medicine. Dept. of Environmental Biology, Okazaki Inst. for Integrative Bioscience

Ca²⁺ measurement using YellowCameleon 3.6 (optical block: DM 510 nm)

The images to the left are an example of ratio imaging. Separated CFP and YFP (FRET) are measured with dual CCD devices. This sequence observed the YellowCameleon 3.6 (Ca²⁺ sensor based on CFP-YFP FRET) expressed Ins-1 cell response with a depolarizing stimulus.

Sample: Ins-1 cell
(insulin-producing cell)
ORCA-D2 optical block: A11400-03 (DM 510 nm, Em1 483 nm/32 nm, Em2 542 nm/27 nm)
Microscope: Olympus IX71
Objective lens: Olympus LUCPlanFLN 60x, NA 0.70

Samples courtesy of Hideo Mogami, Ph.D. Dept. of Physiology, Hamamatsu University School of Medicine. Dept. of Environmental Biology, Okazaki Inst. for Integrative Bioscience

ORCA-D2 compared to conventional CCD image sensor

The low light mode image (ORCA-D2) compared to a conventional high-sensitivity CCD device (ICX285) which is used for scientific ccd cameras.

The brightness profile of the
area shown in the graph
is marked with white horizontal line
on the sample image.

Hamamatsu Photonics K.K. (Japan) is a leading manufacturer of devices for the generation and measurement of UV, visible, and infrared light. These devices include photomultiplier tubes, photodiodes, image sensors, scientific CCD cameras, and light sources. Hamamatsu also manufactures a variety of X-ray solutions as well as specialized photonic systems. We are committed to the advancement of photonics through extensive research.