Spectroscopy Solutions - Random Access Monochromator – DeltaRAM

DeltaRAM X Millisecond Switching Fluorescence Microscope Illuminator

High-speed wavelength switching monochromator for radiometric measurements

  • Millisecond galvanometer switching microscope illuminator
  • Tunable wavelengths anywhere from 250 to 650 nm
  • Continuously adjustable bandpass
  • TTL shutter
  • Couplers for all fluorescence microscopes
  • No alignment required and uniformity assured
Segment: Scientific
Division: Custom Spectroscopy Solutions
Base product
Manufacturing Company: HORIBA Scientific

The ideal fluorescence microscope illuminator for quantitative intracellular ion research of Fura-2 [Ca++], BCECF [pH], SBFI [Na+], FRET and much more

The patented DeltaRAM™ X microscope Illuminator is the ideal fluorescence illuminator for quantitative intracellular ion research. It utilizes a galvanometer based random access monochromator that can switch between any wavelength in 2 milliseconds. The DeltaRAM™ X is a complete, self contained, illuminator that includes a power supply, high intensity xenon light source, DeltaRAM™ X monochromator, TTL shutter and flexible liquid light guide. The wavelength position is a simple analog voltage control.  All you need to add is a microscope adapter for your fluorescence microscope and a USB or PCI DAC interface depending on the software you will be using to drive the illuminator.

The DeltaRAM was developed and patented by Photon Technology International (PTI) with hundreds of installed systems around the world. PTI is the pioneer in the quantitative ratio fluorescence marketplace having introduced the first patented ratio illuminator, the Deltascan™, shortly after the first Fura-2 publication. The DeltaRAM™ X is the newest standard illuminator used with the complete line of PTI EasyRatioPro quantitative fluorescence imaging systems, as well as the PTI RatioMaster™ millisecond PMT photometry system.

The DeltaRAM™ X is widely recognized as the multi-wavelength illuminator of choice with outstanding reliability and customer support.

Compatible DeltaRAM™ X vendor and software platforms

The DeltaRAM™ X can be controlled directly with a variety of third party imaging and instrument software, and HORIBA provides a number of USB and PCI driver interfaces to support these packages.

Better than a Filter Wheel:
Key benefits of the DeltaRAM™ X versus traditional filter wheel illuminators

  • Much faster wavelength switching times
  • Vibration isolation
  • Precise selection of any excitation wavelength and any spectral bandwidth
  • Maximum dynamic range for ratiometric dyes (Fura-2 Rmax/Rmin = 40 with DeltaRAM™ X)
  • Excitation wavelength scanning
  • Much greater value for about the same price

Hardware

The DeltaRAM™ X is a complete self aligned and portable illuminator that includes a flexible 2 meter liquid light guide. There are optional microscope adapters available for virtually any fluorescence microscope. The DeltaRAM™ X can be placed on the microscope bench or on a separate shelf or table top, allowing it to be used outside of a Faraday cage for electrophysiology experiments.

Key Components of the DeltaRAM™ X

  • PowerArc™ xenon arc lamp illuminator and power supply
  • DeltaRAM Random Access Monochromator
  • Liquid light guide
  • Microscope adapter (optional)
  • USB or PCI interface (optional)

PowerArc™ Lamp Housing

The DeltaRAM™ X is powered by a proprietary PowerArc™ lamp housing that requires no cooling or venting. It uses an on-axis ellipsoidal reflector for light collection to collect 70% of the radiant energy from the 75 watt xenon arc lamp compared to only 12%collection efficiency for traditional microscope fluorescence illuminators. The ellipse literally wraps around the arc lamp, collecting 5 to 6 times more output power than from a conventional lamp housing. This outstanding collection efficiency and brightness assures the delivery of as much light as possible through the DeltaRAM™ X random access monochromator. The PowerArc lamp housing is powered by a simple push button, ignition safe, integrated power supply with a lamp usage meter.

Patented DeltaRAM™ X Random Access Monochromator

The heart of the DeltaRAM™ X illuminator is the Random Access Monochromator. The DeltaRAM™ X monochromator is a scanning monochromator that delivers light anywhere from 250 to 650 nm. The monochromator uses a grating to disperse the different wavelengths of light from the incident white light xenon lamp. The scanning element inside the monochromator is a galvanometer which can switch between wavelengths in 2 milliseconds. With a simple and direct voltage control the DeltaRAM monochromator can hop around to multiple wavelength pairs much like a filter wheel. The DeltaRAM™ X however is much faster. It can be continuously tuned to any precise wavelength from 260 to 650 nm and it has an adjustable bandwidth. It can even be scanned like a traditional monochromator to provide spectra.

The DeltaRAM™ X has a continuously adjustable bandwidth because there is no best bandwidth to do any particular fluorescence experiment. Some users prefer to keep the bandwidths as narrow as possible while still getting good throughput because a narrower slit will have less photobleaching. Some experiments however require opening up the slits to maximize speed or signal to noise. With ratiometric experiments such as with Fura-2 or BCECF you want to be sure that the bandwidth is not so large that you will get cross talk between the two excitation channels. For this reason the DeltaRAM™ X lets you empirically test and precisely set the best bandwidths for you. The bandwidth is continuously adjustable from 0 to 24 nm with a manual slit adjustment.

DeltaRAM™ X Wavelength Control

The DeltaRAM™ X is operated remotely, so it requires an external interface, or DAC, that is run by software. To control the DeltaRAM™ X wavelength position you need an interface device that provides a voltage to a BNC connector labeled “position” on the unit. The wavelength position is linear with respect to applied voltage as follows.

DeltaRAM Position:

Applied VoltageCorresponding Wavelength 
4.87 volts250 nm 
0 volts450 nm 
-4.87 volts650 nm 

DeltaRAM™ X Shutter Control

There is a BNC connector labeled “shutter” to digitally open and close the shutter. This is a simple solenoid shutter that is not intended for high speed shuttering.

DeltaRAM™ X DAC Interface Options

PCI DAC: The DeltaRAM™ X can be purchased with an optional 16 bit PCI DAC board that is slotted into a computer. A PCI DAC is necessary for millisecond wavelength switching.

USB DAC: HORIBA also offers two low cost optional USB DAC interfaces for slower wavelength switching requirements. One is based on a National Instruments driver and the other uses a Measurements Computing driver.

The USB DAC using the National Instruments driver converts input commands from 0 to +5 volts into +4.87 to -4.87 volts on the Analog Out 1 BNC. As such, input commands from 0 to 5 volts drive the DeltaRAM™ X from 250 to 650 nm. This interface includes BNC connectors for Shutter 1 and 2, Trigger A and B, Analog Out 1 (DeltaRAM™ X Position) and 2.

The USB DAC using the Measurements Computing driver converts input commands from 0 to +5 volts into +4.87 to -4.87 volts on the Analog Out 1 BNC. As such, input commands from 0 to 5 volts drive the DeltaRAM™ X from 250 to 650 nm. This interface includes BNC connectors for Shutter Out, TTL In, TTL Out and Analog Out (DeltaRAM™ X Position)

DAC Software:

ALL DAC interfaces come with a LabVIEW driver and a simple USB 1.1 compatible interface with a very basic control software package that is Windows2000/XP compliant. This simple program will allow you to set and move wavelengths and manually open and close the shutter.

Applications

Featured Applications

Intracellular Calcium

Brian Research, 921(1-2): 1-11, 2001.
Courtesy of Dr. G. Brewer

  • Fura-2/AM loaded neurons
  • Illuminator: PTI DeltaRAM
  • Camera: Sensys CCD
  • Software: PTI ImageMaster™
  • Imaging: Typical Fura-2-fluorescence ratio imaging for intracellular Ca2+ in hippocampal neurons from old rats before NMDA (A) and after NMDA (B), scale values in nM

Single HEK293 Cell Ca2+ Imaging

PNAS 101 (35), 13062-13067. 2004
Courtesy of Dr. S. Chen

  • HEK293 cell loaded with Fura-2.
  • Illuminator: PTI DeltaRAM
  • Software: PTI ImageMaster™
  • In the picture:(A) Single-cell fluorescent Ca2+ images in the presence (Upper) or absence (Lower) of 0.3 mM caffeine at various [Ca2+]o (0-1.0 mM).
    (B) Fura-2 ratios of representative RyR2(wt) cells in the absence (green trace) and presence (blue trace) of 0.3 mM caffeine and a HEK293 parental cell expressing no RyR2 (pink trace)

Dopamine-induced Ca2+ Transients in Rat MSN

J. Biol. Chem.,279 (40), 42082-42094, 2004. 
Courtesy of Dr. I. Bezprozvanny

  • EGFP transfected rat MSN cultures loaded with Fura2/AM
  • Illuminator: PTI DeltaRAM
  • Camera: PTI IC-300
  • Software: PTI ImageMaster™ Pro
  • Fura-2 340/380 nm ratios in rat MSN before (-1 min) and after (0–28 min) application of 400 µM dopamine

Intracellular Calcium in Human Neuronal Cell Cultures

J. of Neuroimmu, 98(2): 185-200, 1999. 
Courtesy of Dr. H. Gendelman

  • Fura-2AM loaded human neurons
  • Illuminator: PTI DeltaRAM
  • Camera: Photometrics CCCD
  • Software: PTI ImageMaster™
  • Imaging: Fura-2 imaging in SDF-1 treated (B) and control (A) neuronal cells. SDF-1 activate intracellular calcium

Mitochondria [Ca2+]m in Single Cells

J. of Neuroimmu, 98(2): 185-200, 1999.
Courtesy of Dr. H. Gendelman
EMBO, 18(1): 96-108,
Courtesy of Dr. Hajnoczky

  • Fura2FF-loaded single permeabilized RBL cells
  • PTI DeltaRAM illuminator
  • Photometrics PXL CCCD camera

Measurement of Extracellular Near-membrane [Ca2+]

J Cell Sci, 2003, 116(pt 8): 1527-38.
Courtesy of Dr. Hofer, A. M

  • Fura-C18-loaded HEK CaR cells
  • Illuminator: PTI DeltaRAM
  • Camera: PTI IC-100
  • Images a–c: ratio images taken at different time points
  • Image d shows fluorescence at 340 nm excitation (510 nm emission) of the same cells

Mechanical Stimulation Increases Ca2+ Waves

Am J Physiol Lung Cell Mol Physiol 280: L221-L228, 2001.
Courtesy of Dr. S. Boitano

  • Fura-2 AM loaded rat alveolar epithelial cells (AECs)
  • Illuminator: PTI DeltaRAM Camera: PTI ICCD camera
  • Software: PTI ImageMaster™
  • Images: A-D: Mechanical stimulation resulted in a Ca2+ wave that averaged slightly over 4 cells
  • E–H: in the presence of the gap junction-inhibiting peptide Gap 27, [Ca2+]i increase restricted to the stimulated cell
  • I–L: Apyrase did not significantly reduce Ca2+ wave propagation
  • Arrow: Cell that was briefly stimulated with a glass micropipette
  • White lines, cell borders
  • Color bar, approximate [Ca2+]i

Mechanical Wounding Increases Ca2+ Waves

Am J Physiol Lung Cell Mol Physiol 280: L221-L228, 2001.
Courtesy of Dr. S. Boitano

  • Fura-2 AM loaded rat alveolar epithelial cells (AECs)
  • Illuminator: PTI DeltaRAM
  • Camera: PTI ICCD camera
  • Software: PTI ImageMaster™
  • Images: A–D: mechanical wound-induced Ca2+ waves
  • E–H: gap junction inhibitor does not affect this Ca2+ waves
  • I–L: apyrase restricts this Ca2+ waves
  • Arrow: Cell that was mechanically wounded
  • White lines, cell borders
  • Color bar, approximate [Ca2+]i

Intracellular Ca2+ Concentration of ROG Cells in Response to FSH and ATP

Endocrinology Vol. 141, No. 9 3461-3470. 
Courtesy of Dr. T. Ji

  • Fura-2 AM loaded ROG cells
  • Illuminator: PTI DeltaRAM
  • Camera: ICCD camera
  • Software: PTI ImageMaster™

[Ca2+]i Response to ATP

Am J Physiol Lung Cell Mol Physiol 280: L221-L228, 2001.
Courtesy of Dr. S. Boitano

  • Fura-2/AM loaded ROG cells
  • Illuminator: PTI DeltaRAM
  • Camera: ICCD camera
  • Software: PTI ImageMaster™

Regenerative Calcium Oscillations

Biophys J, 79 (5): 2509-2525, 2000. 
Courtesy of Dr. I, Pessah

  • Fura-2/AM loaded differentiated 1B5 myotubes
  • Illuminator: PTI DeltaRAM
  • Camera: ICCD 300 camera
  • Software: PTI ImageMaster™
  • Images: (A) Cells stimulated with 3 mM caffeine. After 2 s, a calcium wave begins from a discrete region and spreads across the cell. After ~2 s more, the calcium wave occurs again. (C)The corresponding change in the Fura-2 340/380 ratio (B) Ratio images from the same cell in A stimulated with 40 mM caffeine. Calcium increases globally throughout the cell, and no calcium waves or oscillations are observed. (D) The corresponding change in the Fura-2 340/380 ratio

Simultaneous Measurement of Phagocytosis and [Ca2+]i

J Cell Sci 2003;116:2857-2865.
Courtesy of Dr. Dewitt, S. et al.

  • Illuminator: PTI DeltaRAM
  • Camera: PTI ICCD100
  • Fura-2 labeled human neutrophils were presented with a DCDHF-labelled C3bi-opsonised particle for phagocytosis
  • Images: Phase contract (top) and corresponding fura2 signal (middle). 90 s: micropipette presenting the particle to the cell; 102 s:adhesion of the particle to the cell without Ca2+ signaling; 123 s: formation of the phagocytic cup; 141s: closure of the phagosome 180 s: completion of the event and the return of cytosolic free Ca2+ to baseline

Correlation of Oxidative Activation with Ca2+ and Phagocytosis

J Cell Sci 2003;116:2857-2865.
Courtesy of Dr. Dewitt, S. et al.

  • Illuminator: PTI DeltaRAM
  • Camera: PTI ICCD100
  • Images: Top: Phase contract to show the phagocytic event
  • Middle row: corresponding fura2 signal to show cytosolic free Ca2+ changes.
  • Bottom row: DCDHF fluorescent intensity of the internalized zymosan particle to assess oxidative activity The graph at the bottom shows the complete time course for cytosolic free Ca2+ change (black) and DCDHF intensity (SI) with the point of phagosomal closure marked by the arrow
  • Conclusion: the onset of oxidative activity correlates with the second phase of the Ca2+ signal.

Local Oxidase Activation and Ca2+ Signal Reported by Fura2-dextran Conjugate

J Cell Sci 2003;116:2857-2865.
Courtesy of Dr. Dewitt, S. et al.

  • Illuminator: PTI DeltaRAM
  • Camera: PTI ICCD100
  • The Fura-2 dextran conjugate micro-injected neutrophils was challenged with an opsonised particle
  • Images: Phase contract (top) and corresponding Fura-2 dextran signal (bottom) show the phagocytic cup (270 seconds), phagosome closure (340 seconds) and completion of the Ca2+signal (380 seconds)
  • The graph on the right shows the complete Ca2+ data, with the point of phagosome closure marked by the downward arrow.

Store-Operated Sr2+ Entry

J. Biol. Chem., 278 (43):42427-42434,2003.
Courtesy of Dr. J. Deans

  • Fura-2/AM loaded CHO cells transfected with CD20 or vector
  • System: PTI DeltaRAM based-ImageMaster™ system
  • Images: (A) Treatment of the cells
  • (B) Images at a, b, and c time points:
  • Before store depletion, no difference in base-line fluorescence, and no Sr2+ entry
  • ATP depleted Ca2+ stores and sharply increased [Ca2+]c in both cell lines
  • Subsequent perfusion of Sr2+ induced a large increase in the CD20-transfected but not the control cells

Use of DCDHF as an Oxidative Indicator During Phagocytosisy

J Cell Sci 2003;116:2857-2865.
Courtesy of Dr. Dewitt, S. et al.

  • Illuminator: PTI DeltaRAM
  • Camera: PTI ICCD100
  • Images: (a) DCDHF-conjugated zymosan particles before (left) and after (right) addition of H2O2
  • The traces below show the time courses for the increase in fluorescence with the arrow indicating the addition of H2O2
  • (b) Fluorescence intensity of internalized (arrowed) and adherent (asterisk) DCDHF-conjugated zymosan particles
  • The DCDHF intensity image and the phase contrast image have been superimposed for clarity.

Ca2+ Imaging of Rat Medium Spiny Neurons

Neuron, 39 (7): 227-239, 2003.
Courtesy of Dr. Ilya Bezprozvanny

  • GFP expression S2 cells loaded with Fura-2
  • Illuminator: PTI DeltaRAM
  • Camera: PTI IC-300
  • Software: PTI ImageMaster™ Pro

Dose-dependence of Trehalose Response in S2-Gr5a Cells

PNAS 100 (suppl. 2):14526-14530, 2003.
Courtesy of Dr. Carlson, J

  • GFP expression S2 cells loaded with Fura-2
  • Illuminator: PTI DeltaRAM
  • Camera: PTI IC-200
  • Software: PTI ImageMaster™
  • Images: Upper: Divided panels of S2-Gr5a cells (Left and Center) or negative controls, transfected with GFP vector alone (Right), before and after application of either trehalose (Left and Right) or maltose (Center)
  • Lower: Images of fields of S2-Gr5a cells taken on application of different concentrations of trehalose.

Time Course of Trehalose Response in S2-Gr5a Cells

PNAS 100 (suppl. 2):14526-14530, 2003.
Courtesy of Dr. Carlson, J

  • GFP expression S2 cells loaded with Fura-2
  • Illuminator: PTI DeltaRAM
  • Camera: PTI IC-200
  • Software: PTI ImageMaster™
  • Images: Upper: A series of images of a single fura 2-loaded S2-Gr5a cell, taken at 5 s intervals
  • Lower: A quantitative representation of the response of the same cell. Bar indicates stimulus period.

Cells Expression Redo-GFP

  • Redo-GFP expressing cells
  • PTI DeltaRAM illuminator
  • Roper Sensy camera
  • PTI ImageMaster™ 3.0 software

Visualization of Mitochondria by mitoGFP

EMBO, 18(1): 96-108,
Courtesy of Dr. Hajnoczky

  • mitoGFP transfected intact (A) and permeabilized (B-D) mast cell
  • Cells were also loaded with MitoTracker Red (C) or rhod2/AM (D)
  • PTI DeltaRAM illuminator.
  • Photometrics PXL CCCD camera
  • The green images (left panels) show the distribution of mitoGFP, the red images (middle panels) show the distribution of MitoTracker Red (C) or compartmentalized rhod2 (D). These images are overlaid in the right panels to show the coincidence of the labeled organelles (overlay).

IP3-induced Intracellular [Ca2+]c and Mitochondrial [Ca2+]m Responses

EMBO, 18(1): 96-108,
Courtesy of Dr. Hajnoczky

  • Fura2FF-loaded permeabilized cell
  • PTI DeltaRAM illuminator
  • Photometrics PXL CCCD camera
  • Left: the overlaid images show the distribution of the membrane-bound CaGreen-C18 (image i, purple) and the mitochondrially compartmentalized Fura2FF (image i, green), and the changes in the Fura2FF fluorescence (images ii-v, 380 nm green/340 nm red) upon addition of 100 nM IP3 (ii versus iii), 12.5 M IP3 (iii versus iv) and ionomycin (iv versus v)
  • Right: time courses of the global [Ca2+]pm response (vi) and the average [Ca2+]m response (vii, thick line), and the [Ca2+]m responses of the marked (1–6 on image i) individual mitochondria (vii, thin lines).

Simultaneous measurement of Ca2+ and myocyte cell length

 

Dual emission channel OBB photometer for Indo-1 Ca++ measurements

The above data traces are from the simultaneous collection of fluorescence and cell length from a cardiac myocyte. Myocyte was loaded with dual emission ratiometric fluorescence probe Indo-1. The OBB/PTI DeltaRAM illuminator was used for excitation illumination at 365 nm and the emitted fluorescence was detected with a dual emission PMT photometer. Above data was collected with FeliX32 photometry software from Photon Technology International (PTI). The blue trace shows the calcium ratio increase and decrease with the cell contraction. The contraction data (Green trace) was detected with a video edge detection electronics module and is correlated with the fluorescence data since both signals were collect simultaneously

Simultaneous Ca++ Fluorescence and Patch Clamp Electrophysiology Research

Image courtesy of, Prof. Dr. György Panyi, Department of Biophysics and Cell Biology, University of Debrecen

Shown is a picture of a PTI/OBB dual emission photometer attached to the C-mount of an inverted fluorescence microscope. The microscope is also equipped with electrophysiology recording devices and perfusion apparatus. The entire setup is inside a Faraday cage for electrical isolation.

OBB’s photometers are ideal for quantitative live cell measurements of intracellular ion and molecules such as Ca++, Na++, pH, GFP, FRET and FRAP experiments. By allowing for simultaneous quantitative fluorescence detection with patch clamp recordings, these photometers have been widely used by electrophysiologists around the world. The OBB PMT photometer is a passive detection system that provides an output voltage signal proportional to the ion/fluorophore of interest, and this signal is directly fed into the electrophysiology A/D converter and collected with the customer’s existing software. The OBB photometer can collect a fluorescence signal at up to 20 KHz for high speed transient recordings.

 

 

Excitation Wavelength Range250–650 nm
Wavelength Selection Speed< 2 milliseconds
Beam Uniformity< 5%
Stray-light rejection10-4
Wavelength Accuracy+/- 1 nm
Optical Output15 mW of optical output in 10 nm bandwidth
Wavelength BandwidthAdjustable from 0–24 nm (manual adjust)
Microscope CouplingTwo meter Liquid Light guide included, and optional adaptors for user specified fluorescence microscope (either direct or epi port coupled)
Wavelength ControlBNC control voltage 
Shutter ControlBNC TTL
DeltaRAM X Specification Sheet
CategoryDocuments
Size 0.89 MB
FiletypePDF
DeltaRAM X App Note Advantage of Using a Scanning Monochromator for Ratio Fluorometric Experiments
CategoryDocuments
Size 0.45 MB
FiletypePDF

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