Microscopes and imaging systems

Manual imaging of multiplex IHC specimens was performed on an Olympus BX-51 microscope (Olympus, Waltham, MA) fitted with a CoolSNAP ES2 CCD camera with 1392 × 1040 pixels and 12-bit resolution (Teledyne Photometrics, Tucson, AZ) and Olympus UPlanSApo 20× (NA 0.75) and 10× (NA 0.40) air objectives. Illumination was provided by either optical filtering of a continuous light source or a 12-LED illuminator. For the former, a Sutter Lambda 10–3 10-position filter wheel (Sutter Instruments, Novato, CA) was used with an Olympus 100 W tungsten halogen lamp to define up to nine wavelength channels. Spectral characteristics (center transmission wavelength and FWHM) of six filters mounted in the filter wheel are listed in Table 1. The relative power of the tungsten lamp and each filtered light channel are plotted as a function of wavelength in Fig. 3a. The LED illumination comprised two Lumencor Spectra X light engines (Lumencor Inc, Beaverton, OR), each containing six custom-selected LEDs, the outputs of which were combined with dichroic beam splitters and focused onto a 3 mm diameter liquid light guide. The 3 mm guides from each light engine were combined into a single 3 mm diameter liquid light guide using a Lumencor combiner, and the light guide connected to the illumination port of the microscope through a Lumencor collimator/microscope adapter. To reduce further the illumination bandwidth, each LED was filtered with a single bandpass optical filter, the spectral characteristics of which are listed in Table 2. The relative output power of each LED versus wavelength is plotted in Fig. 3b, c for unfiltered and filtered LED outputs, respectively. Bandpass filters used with the LEDs and the filter wheel were obtained from Chroma Technology (Bellows Falls, VT) and IDEX Health and Science, LLC (Rochester, NY). Micromanager software was used to control acquisition of the CCD images of individual microscope fields [10].

Normalized extinction coefficients for various chromogens determined for each light channel plotted in Fig. 3a, using filtered tungsten lamp illumination. Coefficients are scaled to 1.000 at the maximum absorbing light channel for each chromogen. Each light channel is designated by the center wavelength of the single bandpass filter and the filter FWHM.

a Illumination spectra of 100 W tungsten microscope lamp (solid black line) in addition to individual light channels produced after bandpass filtering. b Illumination spectra of 12-LED illuminator without filtering. c Illumination spectra of 12-LED illuminator after bandpass filtering of each LED.

Normalized extinction coefficients for various chromogens determined for each light channel plotted in Fig. 3c, using filtered LED illumination. Coefficients are scaled to 1.000 at the maximum absorbing light channel for each chromogen. Each light channel is designated by the center wavelength of the single bandpass filter and the filter FWHM.

The automated pulsed illumination microscope system was constructed by retrofitting a Zeiss AXIO microscope (White Plains, NY) with the custom 12-LED illumination source described above and a Falcon2 8 MPx CMOS monochrome camera operating with 8 bits of resolution and 3328 by 2502 pixels (Teledyne DALSA, Waterloo, ON, Canada). The microscope utilized Zeiss Plan-APOCHROMAT 20× (NA 0.8) and 10× (NA 0.45) air objectives. The complete system was controlled using custom-developed hardware to enable rapid and repeatable image capture. The electronics to control the illumination pulses consisted of an Arduino Mega 2560, which connected to a PC that ran the user interface and camera image acquisition on one side, and discrete TTL logic that provided input/output line buffering for protection of the Arduino circuitry. The PC communicated the exposure times and wavelength sequence to the Arduino via USB communication and the Arduino then controlled the critical timing sequence between the Lumencor illuminators and the monochrome CMOS detector autonomously while the PC processed the images received through the framegrabber card (Dalsa Xcelera-CL PX4 Dual) in parallel. The automated system was capable of acquiring images of a microscope field illuminated sequentially with all 12 LEDs and transferred to the computer in ~700 ms.