Generation 3 programmable array microscope (PAM) for high speed large format optical sectioning in fluorescence

Abstract

We report on the current version of the optical sectioning programmable array microscope (PAM) implemented with a single digital micro-mirror device (DMD) spatial light modulator utilized as a mask in both the fluorescence excitation and emission paths. The PAM incorporates structured illumination and structured detection operating in synchrony. A sequence of binary patterns of excitation light in high definition format (19201080 elements) is projected into the focal plane of the microscope at the 18 kHz binary frame rate of the Texas Instruments 1080p DMD. The resulting fluorescent emission is captured as two distinct signals: conjugate (c, ca. "on-focus") consisting of light impinging on and deviated from the "on" elements of the DMD, and the non-conjugate (nc, ca. "out-of-focus") light falling on and deviated from the "off" elements. The two distinct, deflected beams are optically filtered and detected either by two individual cameras or captured as adjacent images on a single camera after traversing an image combiner. The sectioned image is gained from a subtraction of the nc image from the c image, weighted in accordance with the pattern(s) used for illumination and detection and the relative exposure times of the cameras. The widefield image is given by the sum of the c and nc images. This procedure allows a high duty cycle (typically 25-50%) of on-elements in the excitation patterns and thus functions with low light intensities, preventing saturation and minimizing photobleaching of sensitive fluorophores. The corresponding acquisition speed is also very high, limited only by the bandwidth of the camera(s) (100 fps full frame with the sCMOS camera in current use) and the optical power of the light source (lasers, large area LEDs). In contrast to the static patterns typical of SIM systems, the programmable array allows optimization of the patterns (duty cycle, feature size and distribution), thus enabling a wide range of applications, ranging from patterned photobleaching, (e.g., FRAP, FLIP) and photoactivation, spatial superresolution, automated adaptive tracking and minimization of light exposure (MLE), and photolithography.

Multipoint scanning dual‐detection confocal microscopy for fast 3D volumetric measurement

“…For the MS‐DDCM, light reflected from the sample surface is split by the DMD, and then directed to two charge coupled devices (CCDs). Because in‐focus light passes through the on‐state mirrors and out‐of‐focus light is reflected by the off‐state mirrors, the conjugate and nonconjugate images are measured simultaneously (Heintzmann et al ., ; Jiang & Walker, ; de Vries et al ., ). The conjugate image is analogous to a conventional confocal microscopy image and the nonconjugate image is a widefield microscopy image, minus the confocal image.…”

Sensing Inside the Living Cells

“…The optical sectioning 'intelligent' Programmable Array Microscope (iPAM) can serve as an add-on module for conventional commercial wide fi eld fl uorescence microscope, converting it into a high-speed optical sectioning ("confocal") instrument (de Vries et al 2015 ). It is unique in that it combines both large fi eld of view and high speed, without compromising the imaging performance.…”