Scientific CMOS camera for fast low-light imaging

Hamamatsu has introduced the ORCA-Flash2.8 camera. It is the company's first high-sensitivity digital camera based on a next-generation scientific CMOS image sensor. Designed for low-light imaging at high frame rates, the camera combines high resolution, high sensitivity, high speed, and low noise at an affordable price

The camera's new scientific CMOS image sensor, the FL-280, has 2.8 megapixels and a pixel size of 3.63 x 3.63 µm, ensuring high resolution. The sensor’s wavelength sensitivity ranges from UV to visible, with peak sensitivity (over 60% QE) at about 450-500 nm. The sensor features low readout noise, typically 3 electrons r.m.s. The sensor’s design keeps the readout noise minimal even at very fast readout speeds, unlike traditional image sensors. The FL-280 sensor also has low dark current. Because of its intrinsically low dark current, the sensor requires cooling to +5°C only.

Built for high-speed imaging, the camera's readout speed ranges from 45 frames/second at full resolution up to 1273 frames/second with sub-array readout. Other features include external trigger functions, real-time corrections, and analog gain. It is suitable for a wide variety of applications including ratio imaging, FRET, FISH, TIRF microscopy, and real-time confocal microscopy. The camera will be shown at Analytica 2010 in Munich, Germany, from March 23 to March 26. Demo cameras will be available in May 2010.

More information here.

Improving FRET accuracy with FLIM & environmental control

Mary-Ann Mycek and colleagues from the University of Michigan have used fluorescence lifetime imaging microscopy (FLIM) combined with environmental controls to increase the accuracy of detecting molecular interactions in living cells with FRET. They set out to examine the effect of controlling physiological parameters during FLIM detection of FRET when examining Ras Homology Protein C (RhoC) inactive-form interactions in living cells. This protein has been identified as a specific oncogene marker of aggressive breast cancers.

Using FLIM to detect FRET (rather than intensity-based methods) let the researchers remove intensity-based artifacts, improving fluorescence measurement seven-fold. And using a FLIM system that allowed temperature control and CO2 stabilization eliminated nonspecific FRET in the live cells they studied. The researchers report that the technique, called physiological FLIM, clearly and unambiguously showed the presence of specific molecular interactions in the cells they studied and that it should be generally applicable to various live cell systems.

See other FRET posts including "White light laser allows orange fluorescent protein to be useful acceptor FRET" here.

Research paper:

http://dx.doi.org/10.1117/1.3257254

Ching-Wei Chang, Mei Wu, Sofia D. Merajver, and Mary-Ann Mycek. Physiological fluorescence lifetime imaging microscopy improves Förster resonance energy transfer detection in living cells. J. Biomed. Opt. Vol. 14, 060502 (Nov. 5, 2009)

White light laser allows orange fluorescent protein to be useful FRET acceptor

Ammasi Periasamy of the University of Virginia as well as colleagues from Florida State and Indiana Universities recently studied the performance of an orange fluorescent protein’s performance as an acceptor in a FRET pairs. Orange fluorescent proteins bridge the gap between green and red/far-red fluorescence proteins but are hard to excite with common fixed wavelength lasers. However, the researchers wanted to see if using a supercontinuum light source--also called a white light laser (WWL)--could help. The researchers studied monomeric Kusabira orange 2 (mKO2) as an FRET acceptor partnered with a monomeric teal (mTFP) donor on a standard FRET construct in live cells. They used a supercontinuum laser with continuous emission from 470 to 670 nm that allowed selection of individual wavelengths in 1-nm increments. The researchers found that that mKO2 and mTFP are good FRET partners with the right imaging setup, specifically exciting the mTFP with an Argon 458 nm laser line and using 540-nm excitation from the supercontinuum laser for mKO2.

See other FRET-related posts here.

Research paper:

http://dx.doi.org/10.1117/1.3227036
Yuansheng Sun, Cynthia F. Booker, Sangeeta Kumari, Richard N. Day, Mike Davidson, Ammasi Periasamy, Characterization of an orange acceptor fluorescent protein for sensitized spectral fluorescence resonance energy transfer microscopy using a white-light laser. J. Biomed. Opt. Vol. 14, 054009.

Microscopy camera for bioimaging

QImaging has released the EXi Aqua Bioimaging Microscopy Camera, which replaces the company’s Retiga EXi. The 20 MHz, 14-bit EXi Aqua camera has an IEEE 1394b Firewire interface and achieves mega pixel resolution with a Sony ICX285 interline sensor.

The camera is monochrome but can acquire color with an optional RGB filter module or Bayer mask color CCD option. The camera can read out at up to 10.9 full-resolution frames per second, has flexible binning, and exposure times that are adjustable from 10 microseconds to 17.9 minutes. It is cooled to 0 ºC and exhibits ~20% less read noise than the Retiga EXi. The camera’s QCam driver integrates with a variety of popular third-party software packages.

The camera is useful for many applications, including immunofluorescence imaging, fluorescent protein imaging, time-lapse fluorescence imaging, calcium imaging, and FRET as well as phase-contrast, DIC, and bright field microscopy. More information here.

New CMOS technology for scientific applications

Andor Technology of Belfast, Northern Ireland, Fairchild Imaging of Milpitas, California, USA, and PCO AG of Kelheim, Germany have been working together on a brand new CMOS technology. They introduced the technology by presenting a white paper on the technology at the Laser World of Photonics in Munich on June 16.

According to the companies, the new technology--called scientific CMOS (sCMOS)--will benefit many microscopy applications such as live-cell, super-resolution, and spinning-disk confocal microscopy as well as TIRF, FRAP, FRET, and single molecule detection.

The technology looks to be a significant advancement for biological imaging because it overcomes some of the drawbacks of many sensor technologies, including interline CCDs and electron multiplying (EM) CCDs, which are often used in demanding microscopy applications. sCMOS can simultaneously provide low noise, rapid frame rates, wide dynamic range, high quantum efficiency (QE), high resolution, and a large field of view. 

Here are the numbers for the first sCMOS sensor:

• Sensor format: 5.5 megapixels (2560(h) x 2160(v))
  
• Read noise: <> 16,000:1 (@ 30 frames/s)
  
• QEmax.: 60%
  
• Read out modes: Rolling and Global shutter (user selectable)
  

The white paper provides data and images comparing the sCMOS technology with EM and Interline CCDs. All three companies plan to have cameras using the new technology in 2010, some as early as first quarter.

Get more information on the sensor technology and the white paper at www.scmos.com.

More on this development in the blog post: Is the sCMOS too good to be true? 

Printed FRET pairs

After reading the headline you may be asking why someone would want to print FRET pairs. After all, FRET is typically used to study dynamic protein interactions. Biological applications are indeed what FRET is known for, however scientists are beginning to see that fluorescent proteins might be useful in fabricated devices such as those used for high-density optical memory and switches. If so, FRET could be a useful way to add functionality to devices making use of fluorescent proteins.

To study this possibility, Vinod Subramaniam and colleagues at the University of Twente in The Netherlands printed 2-D and 3-D patterns of fluorescent protein FRET donor/acceptor pairs. The work is detailed in a recent Langmuir research paper.

By examining the spectra and lifetime of the fluorescent proteins using wide-field fluorescence and confocal microscopy the researchers found that FRET occurred at the interfaces between the donor and acceptor molecules. The researchers point out that fluorescent lifetime imaging microscopy (FLIM) is useful to in making sure the function of the patterned fluorescent proteins is not compromised.

In the future, patterning of FRET pairs could be combined with nanopatterning technology to produce nanoscale devices.

Research paper: FRET Pair Printing of Fluorescent Proteins, Maryana Escalante, Christian Blum, Yanina Cesa, Cees Otto, Vinod Subramaniam, Langmuir Article ASAP