PRISM is the widefield multiplane, multicolour super-resolution platform of the Grussmayer Lab at TU Delft. It follows the foundational study on correlative super-resolution fluorescence and multiplane quantitative phase microscopy, and is built as a workhorse instrument: one sample, one illumination arm, and several exchangeable detection arms that can be addressed without realigning the microscope.

On the excitation side four continuous-wave lasers covering the standard wavelengths for biological applications (405 nm, 488 nm, 561 nm, 642 nm) plus an infrared diode laser (830 nm) for the focus lock are mounted. Three illumination geometries are available: (I) a dynamically adjustable dichroic-based Epi-TIRF path with refractive beam shaping, (II) a micro-mirror based objective TIRF path, and (III) a manually positionable LED-based Koehler transillumination for brightfield and quantitative phase imaging. Emission is directed into one of three detection arms: (1) an image-splitting prism producing eight simultaneous focal planes on two sCMOS cameras, (2) a 2D spectral splitter for robust simultaneous multicolour imaging, and (3) an event-based sensor (EBS) arm for high-speed, sparse-signal imaging.

The implementation used in the lab is based on an RM21 body (Mad City Labs, US), which is commercially licensed and can therefore not be shown in CAD; reduced representations of the body are used in the figures below. The PRISM detection concept is not restricted to this body and can be implemented in essentially any widefield, camera-based configuration. A summary of the chromatic properties of the current configuration is available at FPbase. Devices are controlled through micro-manager 2, with acquisitions either set manually, scripted in beanshell, or interfaced through pycro-manager.

System overview

Main body

The objective, the sample stages (also Mad City Labs) and the dichroic filter cube outlined here are mounted in the RM21 body. The body carries the Koehler transillumination on 95 mm rails above the sample, the sample and objective positioners, and the emission splitting dichroic mount that feeds the detection breadboard.

id	Title	Description	Image	Notes	Costs/Supplier
1	RM21 Advanced	Precision aligned Epi-flourescence microscope platform	RM21 advanced	link	Costs/Mad City Labs
2	MicroStage Series	Large (micron - cm) displacement, precision, stepper motor driven, micropositioning system for inverted optical microscopes	Micro-stage	Addressed as MicroDrive XY Stage and MicroDrive Z Stage in micro-manager	Costs/Mad City Labs
3	NanoStage Series	Closed-loop piezo z-positioner used for z-stacks, multiplane calibration and as the actuator of the focus lock		Addressed as MCL NanoDrive Z Stage in micro-manager	Costs/Mad City Labs
4	TIRF-module	Precision aligned linearly translatable mirror-lens configuration to control the angle of illumination and the focusing of the excitation beam onto the back focal plane of the objective lens.	TIRF-module	Continuous transition between epi, HILO and TIRF illumination	Mad City Labs
5	Micro-mirror	Two broadband micro-mirrors positioned at the back aperture of the objective lens.	Broadband micro-mirrors for TIRF imaging and TIRF-lock simultaneous to dichroic based Epi-TIRF	Broadband micro-mirrors for TIRF imaging and TIRF-lock simultaneous to dichroic based Epi-TIRF	Mad City Labs
6	Notch-filter	F40-072 Quad-Notch 400-410/488/561/631-640, mounted in a cage plate.		Placed in the emission path behind the tube lens, blocking all four excitation lines ahead of the splitting optics	AHF, Semrock/ 1100€
7	Objective(s)	60X Super-Apochromate Olympus immersion medium (silicone oil, water) UPLSAPO-S/UPLSAPO-W and oil immersion UPLAPO60XOHR	60x high NA immersion objectives	Silicone and water immersion for live-cell volumes, oil immersion for single-molecule work close to the coverslip	Olympus/
8	Dichroic mirror	ZT405/488/561/640rpcv2-UF2 mounted in magnetic Kinematic Fluorescence Filter Cube DFM1/M	TL dichroic mirror	Ultraflat version to preserve the wavefront across the full field	Chroma/600€ Thorlabs/350€
9	Tube lens	Thorlabs TTL200-A f = 200 mm tube lens		With the 60× objectives this yields 108 nm sampling on 6.5 µm pixels. Mounting is detailed in the detection front end table	Thorlabs
10	Environmental chamber	Cage incubator enclosure covering the stage and trans-illuminatsion, interfacing with the 75mm rails. Edges are sealed with tape, space inbetween breadbord and body is sealed with a folded rubber sheet. Tubing interfaces are designed according to oko-lab specifications. Two doors back and front-side are fixed to the body with M6 threads and knobs.	Environmental chamber designed in SolidEdge	Design and assembly are documented on the live-cell incubator page	self made
11	Environmental control	Oko-lab bold line temperature and CO2 control		Setpoints of 37 °C and 5 % CO2 are reached and held within about an hour after closing the enclosure	Okolabs/
12	Koehler-illumination	OLY-TRANS-ILLUM transillumination kit based upon Olympus IX2-LWUCD condenser suspended on a XT95SD-250 95 mm one-sided rail with XT95RC4/M rail carriers, fixed to XE50L1/M 50 mm square construction rails	Koehler illumination based on Olympus IX2-LWUCD condenser	Construction rails are cut to 300mm height and fixed to the breadboard above the stage with right angle brackets	ASI/ Thorlabs/
13	Trans-illumination stops and filter wheel	Field stop, two filter wheels. One in the LED transillumination arm above the condenser, used to set Koehler conditions and to insert neutral density or chromatic filters for brightfield and quantitative phase imaging. The other an emission path filter wheel which has been removed by now due to the split detection paths, but can be implemented per chromatic path for computational control.		Emission path filter wheel based on the Ries lab 3D printed design	self made
14	Sample positioning	Long-travel xy stage, short-travel xyz stage and a manual z-stage		Long-travel xy = MicroStage, short-travel xyz = NanoStage (rows 2 and 3); manual axial sample positioner	Mad City Labs/
15	Optical table	Thorlabs T1530D Nexus optical table, 1.5 m × 3 m × 310 mm, M6 mounting holes		Carries body, illumination and detection breadboards	Thorlabs

The enclosure is a workshop-manufactured PMMA box (drawing set GELD010084) that seals against the breadboard and the 75 mm construction rails. Its bill of materials is reproduced below; the individual part drawings and the STEP geometry are linked under design files.

Excitation

Four visible excitation lasers with comparably high power levels (>500 mW for the imaging lines) are deployed. The lines are individually expanded, co-aligned on dichroic mirrors and split into two illumination paths by a flip mirror. The main path passes the refractive beam shaper and enters the TIRF module for dichroic-based Epi-TIRF; the secondary path is routed via height-separated periscopes to the micro-mirror TIRF configuration. All optomechanics are mounted on 12 mm optical posts and post holders (Thorlabs) at a beam height of 126 mm.

Beam shaping matters for quantitative single-molecule work: the Gaussian profile delivered by the expanders is converted into a flat top, which widens the usable field, equalises the switching rate across it and removes the intensity gradient that otherwise biases localisation statistics. Behind the shaper two switchable telescopes (2× and 0.67×) set the diameter of the illuminated field, trading field size against intensity, and a flip mirror sends the beam to a monitoring camera for the profile measurements shown below.

Detection

The emission is collected by the objective, passes the tube lens and a shared square field stop, and is then directed by a flip mirror (M_ch) into one of the detection arms. Each arm is built as a 4f relay so that the field stop is imaged onto the sensor and the splitting element sits close to a pupil plane.

id	Title	Description	Image	Notes	Costs/Supplier
1	Emission tube lens	Thorlabs TTL200-A f = 200 mm tube lens in a CXY2A 60 mm cage XY translating mount, carried by LCP33/M and LCP34/M cage plates on ER cage rods and PH50/M post holders		Forms the primary image together with the objective; sets the 108 nm sampling	Thorlabs
2	Quad notch filter	Quad-notch 400-410/488/561/631-640 emission filter in the collimated space behind the tube lens (see the body table, row 6)		Blocks the four excitation lines ahead of the splitting optics	AHF, Semrock/ 1100€
3	Field stop	SP 60 square aperture with four separately positionable edges, mounted on a BA1S/M base with a clamping pedestal at the primary image	Square aperture with 4 separately positionable edges	max. 12 x 12 mm, shared between the detection arms; has to be set manually to the preset field of view of the selected arm	Owis/ 450€
4	Path selection mirror Mch	Broadband dielectric mirror (BB1-E02) in a KMS/M kinematic mount on RS-series pillar posts, seated on a KBM1/M kinematic magnetic base		The magnetic base allows switching between the detection arms and returning to the aligned position without realignment	Thorlabs
5	Detection breadboard	Thorlabs MB3030/M 300 × 300 mm aluminium breadboard carrying the splitter and the cameras, raised on RS-series pillar posts		Keeps the whole splitter arm as one transportable, pre-aligned unit	Thorlabs
6	Light shielding	Enclosure separating the two detection arms and shielding the sensors from stray room light		Self made, based on blackout metal and cardboard sheets sliding into 25mm construction rails, modelled by workshop. Walls and illumination side cover made from anodized aluminium with small cutouts for cable management. Detection side top cover made from blackout cardboard (href="https://www.thorlabs.com/item/TB4">TB4) for simplified adjustments (needs to be cleaned for splinters after cutting).	self made

Multiplane splitter

The multiplane arm images eight focal planes simultaneously. An image-splitting prism with three totally internally reflecting faces divides the beam into eight paths of successively increasing optical path length; four of these are relayed onto camera 1 and four onto camera 2, so that each sensor records a row of four laterally separated sub-images. The optical path increment d between neighbouring paths translates into an axial plane spacing of d/(n·m_t) in sample space, set by the refractive index of the prism glass and the transverse magnification of the relay. The prism used here measures 59.3 mm × 27.3 mm.

The arm can be operated monochromatically, or with an optional spectral-unmixing insert that splits the field into two colour rows ahead of the prism: a longpass dichroic separates the emission, each arm is filtered by its own bandpass, and the two beams are recombined at slightly different angles (±θ) so that they land as two vertically offset rows on each camera. Combined with the eight planes this gives up to sixteen sub-images. An alternative, simpler four-plane arrangement based on a 50:50 beamsplitter and a right-angle prism (plane spacing L/2n) is shown in the same figure for setups that do not need eight planes.

Ahead of the prism a set of kinematic folding mirrors introduces small, adjustable path length offsets between the sub-beams (+3 mm and −2 mm in the current configuration), which shifts the plane spacing without exchanging the prism. Plane calibration is then performed on fluorescent beads: a z-stack through the sample yields the brightness profile of every sub-image, and the offsets between the profile maxima give the axial position of each plane as well as the inter-plane distances used in the reconstruction.

id	Title	Description	Image	Notes	Costs/Supplier
1	Fourier lens Fm,R1	#49-366-INK 25mm Dia. x 250mm FL, VIS-NIR, Inked, Achromatic Lens (400-1000nm)		Collimates the intermediate image relayed from the field stop	Edmund optics/ 120€
2	Fourier lens Fm,R2	#49-365-INK 25mm Dia. x 225mm FL, VIS-NIR, Inked, Achromatic Lens (400-1000nm)		Sets the transverse magnification mt of the relay and with it the axial plane spacing	Edmund optics/ 120€
3	Beam deflection mirror	KCB1/M mount with broadband dielectric mirror (BB1-E02)	KCB1/M	Inserted due to spatial constraints	Thorlabs 300€
4	Multiplane-splitter	Image splitting prism with three TIR reflectors, 59.3 mm × 27.3 mm, distributing eight optical paths over two cameras	Image splitting prism, eight paths distributed over two cameras	Allows simultaneous detection of diffraction limited images at equally spaced axial planes with only minimal alignment of the overall optical setup. Assembly from off-the-shelf beamsplitters is documented on the prism fabrication page	Scoptonic Imaging technologies/10-20k€
5	Multiplane-splitter-mount	Multi-plane prism aligner	Multi-plane prism aligner, Scoptonic Imaging technologies	Multi-Axis stage: X, Y, Z, Pitch, Yaw and Roll adjustment	Scoptonic Imaging technologies
6	Spectral unmixing insert	Longpass dichroic, two bandpass filters and three folding mirrors placed at 112.5 mm / 115.0 mm / 110.0 mm that recombine two colour channels under ±θ		Optional; splits every plane into two vertically offset colour rows on the sensor	Thorlabs, Semrock/
7	2x sCMOs cameras	Teledyne Photometrics KINETIX	Teledyne Photometrics KINETIX	3200 x 3200 pixel, air/water cooled, 83Hz full range full chip, high sensitivity sCMOS; addressed as a MultiCamera device through PVCAM	Teledyne Photometrics
8	Path length trim mirrors	Kinematic folding mirrors (labelled I, IIa,b, III in the schematic) that add the +3 mm and −2 mm optical path offsets between the sub-beams before the prism		Used to fine-tune the interplane spacing without exchanging the prism; mirror substrates BB1-E02 in KM100CP/M and KMS/M mounts	Thorlabs
9	Prism mount and translation	Prism aligner on a KM200B/M kinematic mount with an XYT1/M translation mount, bolted to the MB3030/M detection breadboard		Alternative off the shelf prism mount, the Scoptonic six-axis aligner in row 5 is the main mounting option	Thorlabs
10	Camera mounting adapter	Machined adapter plate coupling the Kinetix housing to the breadboard, setting sensor height (112.5mm center)and orthogonality with respect to the prism	Camera translation stage adapter	Self made (kinetix_to_plate_adapter)

2D spectral splitter

The second detection arm splits the field into up to four spectral channels that are tiled onto a single sCMOS sensor, which makes simultaneous multicolour imaging robust against sample motion and against bleaching between sequential colour frames. The channels are separated by ultraflat dichroics in a mono-to-quad view splitting platform and re-imaged by a common Fourier lens pair; the shared square field stop with four independently positionable edges defines the tile size.

Channel registration and the point spread function of each channel are calibrated on beads. The measured PSF widths of the two main channels agree to within a few percent laterally (about 0.37–0.40 µm) and axially (about 0.95–0.97 µm), which is what allows localisations from different channels to be combined into a single, spectrally demixed reconstruction.

id	Title	Description	Image	Notes	Costs/Supplier
1	Path separation mirror Mch	Kinematic mirror mount on magnetic base for path separation		The magnetic base allows switching between detection arms without realignment	Mad City Labs/ Thorlabs/
2	MadView multiview beam splitting platform	Mono-to-quad view adaptable beam-splitting system designed for multi-color Single Molecule Microscopy imaging.			Mad City Labs/
3	Dichroic mirror mounts	6x mounts (2 per split) for 26x38x3mm ultraflat dichroics (λ/4 Peak-to-Valley flatness)		Flatness is critical to avoid channel-dependent aberrations	Thorlabs/
4	Fourier lenses Fci,R1	225mm focal length collimation lenses conjugate to the tube lens mounted in SM1L03 lens mount			Mad City labs /
5	Focussing lens	#49-364-INK 25mm Dia. x 200mm FL, VIS-NIR, Inked, Achromatic Lens (400-1000nm)			Edmund optics/ 120€
6	sCMOs camera	Teledyne Photometrics BSI Express	Teledyne Photometrics BSI Express	2048 x 2048 pixel, air cooled, 43Hz full range full chip, high sensitivity sCMOS	Teledyne Photometrics
7	Aperture / field stop	SP 60 square aperture with 4 separately positionable edges	Square aperture with 4 separately positionable edges	max. 12 x 12 mm, shared among both detection paths (needs to be manually adjusted to preset FOVs)	Owis/ 450€
8	Emission filters	One bandpass filter per spectral channel behind the splitting dichroics		Filter set follows the FPbase configuration	AHF, Semrock
9	Folding mirrors M1, M2	Kinematically mounted broadband dielectric mirrors that fold the split channels onto their tiles on the sensor			Thorlabs

Event-based sensor path

A third detection arm carries an event-based sensor (EBS). Instead of integrating frames, every pixel reports asynchronous ON/OFF events whenever the logarithm of the incident intensity crosses a threshold, which gives microsecond temporal resolution at low data rates for sparse, moving signals. On PRISM the arm shares the splitter path through a 50:50 beamsplitter, so the same field can be recorded on the sCMOS and on the EBS at the same time. That makes the arm useful for fast single-particle tracking, where the frame rate of a conventional camera limits the accessible dynamics.

Focus lock

Axial drift is suppressed by an infrared reflection focus lock. An 830 nm collimated diode beam is coupled in through the micro-mirror path, totally internally reflected at the sample coverslip and imaged onto a quadrant photodiode; the lateral position of the reflected spot on the quadrants is proportional to the axial displacement of the coverslip and is fed back onto the piezo z-stage. Readout and feedback are handled by the Mad City Labs TIRF Lock, which closes the loop in software onto the nanopositioner. Because the lock operates beyond the emission bands and enters through the micro-mirror path, it can run simultaneously with dichroic-based Epi-TIRF imaging.

The benefit shows in long single-molecule acquisitions: with the lock engaged the mean axial position of the localisations stays flat over tens of thousands of frames, while it drifts continuously when the lock is off — in particular with the incubator running, which is exactly the regime needed for live-cell measurements.

Control and acquisition

All devices are addressed through micro-manager 2: the two Kinetix cameras through PVCAM as a combined MultiCamera device, the BSI Express on the splitter path, the Mad City Labs MicroDrive XY and Z stages and the NanoDrive piezo, the laser heads over their serial interfaces, and the shutters and line switching through an Arduino hub. Acquisition routines are written against pycro-manager, which allows the acquisition engine to be extended with per-frame image processors and hooks.

Three routines carry most of the day-to-day work: an on-the-fly processor that regroups the interleaved multi-camera stream into per-plane TIFF files with matching metadata while the acquisition is running; an unattended multi-position routine that autofocuses, segments the brightfield channel with Cellpose and only stores fields that actually contain cells; and a closed-loop STORM controller that estimates the density of active emitters per frame and adjusts the 405 nm reactivation power through a PI controller to hold the blinking density in the optimal range.

Characterisation and performance

The instrument is characterised with a small set of routine measurements: pixel size against a calibration ruler, illumination flatness per line, plane positions from bead stacks, PSF per spectral channel, mechanical and axial stability, and a phase calibration against a step of known height for the quantitative phase modality. The standards and routines used for each are listed below.

Design files and related pages

• Prism fabrication — assembly of multiplane splitters from off-the-shelf beamsplitters.
• Live-cell incubator — the cage incubator design for the RM21 platform.
• Incubator STEP file — enclosure geometry as used on this system.
• FPbase configuration — spectra of the current filter and splitter configuration.
• GrussmayerLab on GitHub — acquisition and processing code for the multiplane pipeline.

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