Fluorescence Spectrophotometer F P- -8000 series 8000 series F P UV/CD Division JASCO Corp 1 1
Introduction of New Instruments JASCO started in 1958 as a company manufacturing IR dispersive spectrophotometers Since then, several other instruments have been developed for commercial sales: UV-Vis(1961) CD/ORD(1964) Polarimeter(1965) Spectrofluorometer(1967) Laser Raman(1969) HPLC(1972) Cell sorter/Flow cytometer(1982) SFC(1985) Cell fusion(1986) Gas permeability tester(1987) Ellipsometer(1989) NUVOM(1991) Ca ion analyzer(1991) Hi-SOR-CD(1998) SNOM(2000) VUV(2000) 2 2
History of JASCO Spectrofluorometers 1976 FP-550 1976 SFP-3-1 1980 FP-550A 1985 FP-770 1989 FP-777 1996 FP-750 1997 FP-715 1967 FP-1 2000 FP-6600, 6500, 1968 FP-2 6300, 6200, 6100 1969 FOM-1 1970 FP-3 1970 FL-10 1971 FOM-2 1972 FP-4 1975 FP-100 3 3
Diverse Demands of the Market High sensitivity Spectra Quantitative Analysis Fluorescent Analysis Europe and US Eastern Asia and Japan Trends Solid Biotech Sample Evaluation of FPD,LED Materials NIR Proteins Emission Spectra Fluorescent Quantum Efficiency Phosphorescence Measurement The fluorescence reagent of deep-red Ex. Carbon nanotube Rare earth element Fluorescent Automatic Titrator High temperature powder cell NIR Fluorescence Micro Plate Measurement Unit. Luminous color Spectrophotometer Automatic Depolarization measurement/analysis program ~ 1400nm measurement Acc. Quantum efficiency measurement ~ 1700nm Stoped Flow Unit unit for solid sample Phosphorescence measurement for solid sample 4 4
Requirements for Life Science Applications � Micro-sampling � Automation (Auto-sampler, Micro-plate reader) � Fluorescence depolarization (anisotropy) � Auto-titration � Stopped flow 5 5
Requirements for Advanced Materials Research � Accurate quantum yield determination � Accurate spectral correction � Solid or powder samples � Micro particle samples � Luminous materials (White LED & …) � Phosphorescence (Organic EL) � Electric-field inducing Abs. (Organic EL) 6 6
Requirements for NIR Fluorescence Mesurements � Carbon nano-tube analysis • Chirality determination of CNT � NIR & deep red fluorescent dye • High sensitivity measurement in crude sample 7 7
Improvement Requests For Next FP Series � Improvement of noise to signal ratio � A precise excitation light monitor � Improvement in user-friendliness • Suppression high-order diffraction light • Basic and advanced parameters setup • Simple and accurate spectrum correction � Enhanced accessories 8 8
Development Objectives � 4 models: The successors of FP8200/8300/8500/8600. � The fundamental performance of each model’s monochromator will be improved. � High speed excitation shutter. � Auto gain ~ Expand dynamic range. � Spectra Manager 2/CFR, Automatic accessories recognition, USB2.0 for data communication. � Enhanced accessories. � iRM with color LCD 9 9
Instrument Outline ➢ Series products by function, performance & applications ➢ Enhancements for performance, S/N, speed, dynamic range, etc. ➢ Improvement in optics and signal processing ➢ Artifact free spectra by higher-order diffraction cut-filters, accurate Excitation monitoring, etc. ➢ Instrument appearance 10 10
FP-8000 series line FP-8200 High performance routine model - Wide dynamic range over 6 orders of magnitude fluorescence emission - Spectra free from higher order diffraction light (option) FP-8300 Enhanced model for Bio-science - Updated application systems for Bio-analysis applications (Microplate reader, Stopped flow , Fluorescence depolarization, Titration) - Wide dynamic range over 6 orders of magnitude - Spectra free from higher order diffraction light (standard) - Expansion to materials analysis (Phosphorescence, Integrating sphere) FP-8500 Evolved for advanced materials evaluation - Highest sensitivity - Highest scan speed - Wide dynamic range over 6.5 orders of magnitude - Spectra free from higher order diffraction light (standard) FP-8600 For evaluation of new technology materials - Covering NIR region - Highest scan speed 11 - Spectra free from higher order diffraction light (standard) 11
Updated Appearance – Contemporary Design F P-8500 12 12
13 13 P-8500/ 8600 570 F 270 545 Instrument Dimensions 520 P-8300 F 490 P-8200 F
New Functions and Features • High S/N F P-8200/ 8300/ 8500/ 8600 • Wide dynamic range F P-8200/ 8300/ 8500/ 8600 • High scan speed F P-8200/ 8300/ 8500/ 8600 • Improved wavelength resolution F P-8200/ 8300 • Higher order diffraction cut filter F P-8200/ 8300/ 8500/ 8600 • Improved NIR performance F P-8600 • Improved spectral correction F P-8200/ 8300/ 8500/ 8600 • Enhanced phosphorescence F P-8300/ 8500/ 8600 • Non-contact IQ accessory F P-8200/ 8300/ 8500/ 8600 • USB communication F P-8200/ 8300/ 8500/ 8600 • Spectra Manager II & CFR F P-8200/ 8300/ 8500/ 8600 • iRM-900 F P-8200/ 8300 • Updated accessories F P-8200/ 8300/ 8500/ 8600 14 14
Feature 1: Improvement in S/N The most important feature in Fluorescence F P- -8000 8000 F P 1:380 (SBW=5nm) F P-8200 1:680 (SBW=5nm) F P-8300 1:1200 p-p ( SBW= 5nm) F P-8500 1:600 (EM SBW= 10nm) F P-8600 15 15
Feature 1: Improvement in S/N Water Raman [Peak Signal]/[Peak Noise], Ex 350nm FP-8500: >1200 (RMS), >300 (p-p) 1840 Peak to Peak 351.9 Signal = 1910 RMS 1534.1 1820 Int. 1800 1780 0 2 4 6 8 10 Time [sec] • Advanced AD enables rapid sampling at the rate 0.2 μ sec for emission data, and new high speed signal processing by FPGA contributes to considerable improvement of the efficiency for signal acquisition • P-P S/N is calculated according to JIS K0120 16 16
Feature 1: Improvement in S/N Water Raman [Peak Signal]/[Baseline Noise], Ex 350nm FP-8500: >5000 (RMS) Signal = 1910 RMS Noise = 0.2257 S/N = 8463 • S/N ratio at baseline is much larger than at the Raman peak. • Noise evaluated at baseline(450nm) is much smaller than at the Raman peak. • Noise is roughly proportional to signal intensity. 17 17
Feature 2: Expansion of Dynamic Range New Auto-Gain & Auto-SCS which provides a remarkable expansion of fluorescence dynamic range 18 18
Feature 2: Expansion of Dynamic Range 2400 520 AutoGain ON 500 2000 AutoGain OFF 450 Int. Int. 1000 Zoom in 400 0 380 600 620 640 660 680 700 661 662 663 664 665 Wavelength [nm] Wavelength [nm] A single spectrum collection can provide reliable acquisition of a very small peak together with a coexisting large peak. This feature is very useful for the determination of fluorescence quantum yield. 1.2 600 AutoGain ON 1 AutoGain OFF 400 Int. Zoom in the 0.5 Int. foot of the 200 small peak 0 0 520 540 560 580 600 -100 19 300 400 500 600 700 750 Wavelength [nm] Wavelength [nm] 19
Feature 2: Expansion of Dynamic Range Auto-SCS allows measurement from sub-picomol. to micro-mol, without manually changing the instrument “Sensitivity” setting. F P-8200 F P-8500 Fluorescein Fluorescein Density Density 5 × 10 -13 ～ 1.8 × 10 -6 mol/L 5 × 10 -13 ～ 1.5 × 10 -6 mol/L Linearity over 6.5 orders of Linearity over 6 orders of magnitude magnitude log(Y) = A × log(X) + B log(Y) = A × log(X) + B A = 1.02 3 A = 1.00 7 B = 9.85 5 B = 9.85 0 C.C. = 0.9999 C.C. = 0.9999 20 Std.Err.= 5.58 5 E-008 Std.Err. = 3.46 9 E-008 20
Feature 2: Expansion of Dynamic Range Several steps is needed to cover 6 orders in previous model. log I 1 10 100 0.0001 0.01 1 100 x100 x100 log I log I log I 0.01 1 10 100 0.0001 0.001 0.01 0.1 1 21 21
Feature 3: Improvement in Scan Speed F P- -8000 8000 F P 20000nm/min F P-8200 20000nm/min F P-8300 60000nm/min F P-8500 Ex:60000nm/min F P-8600 Em:120000nm/min Advanced sine-bar driving screw and new monochromator motor drive results in a much faster scan speed for all instruments. 22 22
Feature 4: Improvement in Wavelength Resolution F P- -8000 8000 F P 2.5nm F P-8200 1nm F P-8300/ 8500 23 23
Feature 5: Higher Order Diffraction Cut Filters Fluorescence instruments have always Fluorescence instruments have always suffered from higher order diffraction effects. suffered from higher order diffraction effects. Ex spectrum of anthracene Em Spectrum of Orange plate Filter OFF Filter ON 24 24
25 25 (FP-8600 Em only) R-70 Feature 5: Higher Order Diffraction Cut Filters Y-48 WG-305 SiO2
Feature 5: Higher Order Diffraction Cut Filter without cut filters with cut filters The new ‘cut filter’ capability removes peaks originating from higher order diffracted light to provide simple, reliable spectral acquisition and analysis. This system works quite well for 3D fluorescence measurements to reveal peaks usually hidden by the high order diffraction peak. The cut filter method also works quite well for quantum yield determination by providing measurements of accurate spectra. 26 26
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